Effect of Selenium Supplementation from Various Dietary Sources on the Antioxidant and Selenium

Status of Dairy Cows and Trace Element Status in Dairy Herds

D i s s e r t a t i o n

zur Erlangung des akademischen Grades doctor rerum agriculturarum

(Dr. rer. agr.)

eingereicht an der Landwirtschaftlich-Gärtnerischen Fakultät der Humboldt-Universität zu Berlin von Salman Saeed, M.Sc (Hons.) Animal Nutrition aus Pakistan (geboren am 05.11.1974, Lahore)

Präsident

der Humboldt-Universität zu Berlin

Prof. Dr. Dr. h.c. Christoph Markschies

Dekan der

Landwirtschaftlich-Gärtnerischen Fakultät

Prof. Dr. Dr. h.c. Otto Kaufmann

Gutachter: 1. PD Dr. Helmut Schafft

2. Prof. Dr. Jürgen Zentek

Tag der mündlichen Prüfung: 08. 03. 2010

2

In the sweet memories of my loving brother

Abul’Aala Sultan Saeed (May Allah shower His blessings upon him)

who always inspired me towards higher ideals in life

3

ACKNOWLEDGEMENT

All praises be to Allah Almighty who bestowed upon me His uncountable and

immeasurable blessings. And peace be upon His noble messengers, the last of

whom was Prophet Muhammad (SAW), who brought the light of knowledge and

wisdom to the mankind.

Firstly, I am grateful to Dr. Claudia Kijora and Prof. Dr. Ortwin Simon, Director of the

Institute of Animal Nutrition FU, for their kind guidance towards my acceptance as

PhD student. I am heartily thankful to my supervisor, Prof. Dr. Jürgen Zentek, whose

encouragement, guidance and support from the initial to the final level enabled me to

develop an understanding of the subject. I owe my deepest gratitude to Dr. Helmut

Schafft, for co-supervising my project and for especially giving his valuable

suggestions during the process of thesis writing and final submission. This project

would not have been possible without the sincere and kind cooperation extended by

Dr. Monika Lahrssen-Wiederholt, Prof. Dr. Howard Hulan, Dr. Annabella Khol-

Parisini, Mrs. Heide-Marie Lochotzke and many friendly and supportive workers

taking care of the dairy facility at the Bundesinstitut für Risikobewertung (BfR). I owe

my special thanks to Dr. Klaus Schäfer and Dr. Matthias Schreiner, Department of

Food Science and Technology, Universität für Bodenkultur (BOKU), Vienna, for the

valuable guidance and help in establishing the analytical methods. I am indebted to

Prof. Dr. Klaus Männer and Dr. Wilfried Vahjen for their kind help during the whole of

my stay at the institute.

I would like to thank my colleagues Anett Kriesten, Daniela Dinse, Petra Huck, Marita

Eitinger and Sybille Weinholz who have always been there to help me in the labs

whenever I needed. The limitation of space on this page bars me to mention the

4

names; however, I can’t forget the friendly environment offered by all other

colleagues in the institute of animal nutrition.

It would have a dream to complete my studies abroad without the devout prayers of

my parents, family, brother and sisters. Particularly, I feel myself greatly indebted to

my wife and my only son for their support in the form of patience, love and care they

offered to me and the whole family and how they kept the gap of my absence at

home filled.

Lastly, I offer my regards to all of those who supported me in any respect during the

completion of my studies. My friends Hafiz Zahid, Ahsanullah, Hafiz Haroon, Abdul

Jabbar, Usman, Husnain, Rizwan ul Haq, Qasim Mushtaq, Sulaiman Khan, Dr. Abid

Riaz, Adeel Zaffar, Ilyas Sadiq, Shahid Qureshi, Imtiaz Rabbani, Sultan, Haroon-ur-

Rasheed, Imran Gul, Waqas Latif and many others made it possible for me to feel “at

home” in Germany. I am also obliged to Higher Education Commission (HEC) of

Pakistan and German Academic Exchange Service (DAAD) for granting me the

scholarship and my home institution for the study leave. The grant provided by the

Sächsisches Landesamt für Umwelt, Landwirtschaft und Geologie for the survey part

of this work and cooperation from Dr. Olaf Steinhöfel and Mrs. Fröhlich is also highly

acknowledged.

Thanks a lot to all of you!

Salman Saeed

December 18, 2009

5

TABLE OF CONTENTS

LIST OF TABLES ................................................................................................................................................. I�

LIST OF FIGURES .............................................................................................................................................. II�

ABBREVIATIONS ............................................................................................................................................. III�

ZUSAMMENFASSUNG ...................................................................................................................................... V�

ABSTRACT ...................................................................................................................................................... VIII�

1.� INTRODUCTION ......................................................................................................................................... 1�

2.� REVIEW OF LITERATURE ....................................................................................................................... 3�

2.1� SELENIUM: FROM TOXICITY TO ESSENTIALITY .................................................................................... 3�2.2� SELENIUM AND MECHANISM OF OXIDATIVE STRESS .......................................................................... 4�2.3� METABOLISM OF SELENIUM IN MAMMALS ........................................................................................... 7�2.4� SELENIUM NUTRITION OF DAIRY COWS .............................................................................................. 9�2.5� BIOMARKERS FOR SELENIUM STATUS............................................................................................... 10�2.6� SOMATIC CELL COUNT AND SELENIUM STATUS ............................................................................... 13�2.7� MASTITIS SUSCEPTIBILITY AND SELENIUM STATUS .......................................................................... 14�2.8� MAMMARY GLAND IMMUNE SYSTEM – INTERACTIONS WITH SELENIUM ........................................... 15�

2.8.1� Physical Barriers ......................................................................................................................... 16�2.8.2� Cellular Factors ........................................................................................................................... 16�2.8.3� Soluble Factors ........................................................................................................................... 23�

2.9� CONCLUDING REMARKS .................................................................................................................... 28�

3.� MATERIALS AND METHODS ................................................................................................................ 29�

3.1� FEEDS AND ANIMALS ......................................................................................................................... 29�3.2� SAMPLING .......................................................................................................................................... 31�3.3� CHEMICALS AND INSTRUMENTS ......................................................................................................... 31�3.4� ESTIMATION OF SELENIUM ................................................................................................................ 31�3.5� ESTIMATION OF ANTIOXIDANT ACTIVITY ............................................................................................ 32�3.6� STATISTICAL ANALYSIS ...................................................................................................................... 32�

4.� RESULTS ................................................................................................................................................... 33�

4.1� COLOSTRUM AND MILK SELENIUM STATUS ...................................................................................... 33�4.2� MILK TROLOX EQUIVALENT ANTIOXIDANT CAPACITY (TEAC) ......................................................... 34�4.3� MILK PRODUCTION ............................................................................................................................. 35�4.4� SERUM SELENIUM IN COWS .............................................................................................................. 36�4.5� SERUM SELENIUM LEVEL IN CALVES ................................................................................................ 37�4.6� BODY MASS OF CALVES .................................................................................................................... 38�4.7� SERUM TEAC IN COWS..................................................................................................................... 39�4.8� SERUM TEAC IN CALVES .................................................................................................................. 40�

5.� DISCUSSION ............................................................................................................................................. 42�

5.1� COLOSTRUM AND MILK SELENIUM STATUS ...................................................................................... 42�5.2� TOTAL ANTIOXIDANT CAPACITY IN MILK ............................................................................................ 44�5.3� MILK SELENIUM AND TEAC RELATIONSHIP ...................................................................................... 45�5.4� MILK PRODUCTION ............................................................................................................................. 46�5.5� SERUM SELENIUM CONTENT IN COWS .............................................................................................. 47�5.6� SELENIUM TRANSFER FROM COWS TO CALVES ............................................................................... 48�5.7� SERUM TROLOX EQUIVALENT ANTIOXIDANT CAPACITY (TEAC) IN COWS ...................................... 49�5.8� SERUM TEAC IN CALVES .................................................................................................................. 51�

6.� TRACE ELEMENT STATUS IN LARGE DAIRY HERDS ................................................................... 52�

6.1� INTRODUCTION ................................................................................................................................... 52�6.2� FARMS, ANIMALS AND SAMPLING ...................................................................................................... 53�6.3� STATISTICAL ANALYSIS ...................................................................................................................... 54�6.4� RESULTS AND DISCUSSION ............................................................................................................... 54�

6

7.� CONCLUSION ........................................................................................................................................... 65�

8. REFERENCES ................................................................................................................................................. 66�

APPENDIX 1 DATA ON FEEDING TRIAL WITH DAIRY COWS CONDUCTED AT BFR ..................... 83�

APPENDIX 2 SAXONIAN DAIRY HERDS DATA – FEED COMPOSITION ............................................. 85�

APPENDIX 3 SAXONIAN DAIRY HERDS DATA – TRACE ELEMENTS, HEALTH AND PRODUCTION PARAMETERS ........................................................................................................................ 91�

i

LIST OF TABLES

Table 1 Dairy cattle immune responses as affected by selenium ............................ 26�

Table 2 Bovine udder health and mastitis susceptibility as affected by selenium .. 27�

Table 3 Composition of total mixed ration (TMR) fed as basal diet during the

feeding trial ..................................................................................................... 30�

Table 4 Mineral composition (DM basis) of total mixed ration (TMR) fed as basal

diet during the feeding trial (n=3) ................................................................... 30�

Table 5 Milk and nutrients yield in different treatment groups during the feeding

trial .................................................................................................................. 36�

Table 6 Health status of experimental cows during feeding trial ............................ 36�

Table 7 Body mass (kg) of cows and calves in various treatment groups .............. 39�

Table 8 Regression equations describing the relationship between milk TEAC and

selenium levels in various treatment groups ................................................... 46�

Table 9 Regression equations describing the relationship between calves and dams

serum selenium levels in various treatment groups ........................................ 49�

Table 10 Regression equations describing the relationship between serum selenium

and TEAC in dams .......................................................................................... 50�

Table 11 Descriptive summary of feed composition (DM basis) data collected from

11 different farms in Saxonia (Germany) ....................................................... 55�

Table 12 Component matrix resulted from the principal component analysis of feed

data of 11 dairy herds ...................................................................................... 56�

Table 13 Descriptive summaries of various parameters measured in samples

collected from 11 different farms in Saxonia (Germany) ............................... 58�

Table 14 Pearson correlation matrix for various trace elements in liver tissues ..... 59�

Table 15 Regression equations describing relationship between feed and liver

tissues concentrations of various trace elements ............................................. 63�

Table 16 Multiple linear regression models describing the relationship among liver

trace elements (dependent) and feed composition (independent) ................... 63�

Table 17 Multiple linear regression models describing the relationship among liver

trace elements and various parameters of herd performance .......................... 64�

ii

LIST OF FIGURES

Figure 1 The redox states of oxygen with standard reduction potential (volts). ................ 5�

Figure 2 Selenium incorporation in proteins (Suzuki 2005) .............................................. 9�

Figure 3 Selenium metabolism in mammals (Suzuki 2005) .............................................. 9�

Figure 4 Chemical structure of ABTS molecule .............................................................. 32�

Figure 5 Colostrum and milk selenium concentrations in various treatment groups. ...... 34�

Figure 6 Milk TEAC values at various lactation stages in different groups. ................... 35�

Figure 7 Serum selenium concentrations of dams in different groups during various

physiological stages. ................................................................................................. 37�

Figure 8 Serum selenium concentrations of calves borne to dams in different treatment

groups. ...................................................................................................................... 38�

Figure 9 Differences in body mass of calves at various time points ................................ 39�

Figure 10 Serum TEAC values in cows of various treatment groups. ............................. 40�

Figure 11 Serum TEAC in calves of various treatment groups. ...................................... 41�

Figure 12 Overall milk selenium and TEAC regression model. ...................................... 46�

Figure 13 Regression model describing the relationship between dams and calves serum

selenium levels. ........................................................................................................ 48�

Figure 14 Regression model describing the relationship between serum selenium and

TEAC in dams. ......................................................................................................... 50�

Figure 15 Regression model describing the relationship between calves’ serum selenium

and TEAC values. .................................................................................................... 51�

Figure 16 Trace elements results measured compared to a standard ............................... 54�

Figure 17 Screeplot diagram of the feed components ...................................................... 57�

Figure 18 Feed and liver selenium relationship in Saxonian dairy farms ........................ 60�

Figure 19 Feed and liver zinc relationship in Saxonian dairy farms ................................ 61�

Figure 20 Feed and liver copper relationship in Saxonian dairy farms ........................... 61�

Figure 21 Feed and liver iron relationship in Saxonian dairy farms ................................ 62�

Figure 22 Feed and liver manganese relationship in Saxonian dairy farms ..................... 62�

iii

ABBREVIATIONS ADF Acid detergent fiber

DTNB 5,5´-dithio-bis(2-nitrobenzoic acid)

EDTA Ethylene-diamine-tetraacetic acid

FRAP Ferric reducing ability of plasma

GSH Reduced glutathione

GSHPx Glutathione Peroxidase

GSSG Glutathione disulfide

KS Kolmogorov-Smirnov

NADPH Nicotinamide adenine di nucleotide phosphate

NDF Neutral detergent fibre

NK Natural killer

ORAC Oxygen radical absorption capacity

RNS Reactive nitrogen species

ROS Reactive oxygen species

SEM Standard error of mean

SeY Selenium yeast

SeI Selenium inorganic (sodium selenite)

TEAC Trolox equivalent antioxidant capacity

TMR Total mixed ration

TRAP Total peroxyl radical trapping parameter

TROLOX 6-hydroxy-2, 5, 7, 8-tetramethychroman-2-carboxylic acid

iv

Reactive Species

H2O2 Hydrogen peroxide

-OH Hydroxyl radical

NO Nitric oxide

ONOO- Peroxynitrite

O2- Superoxide radical anion

Amino Acid Codes

Asp Aspartate

Cys Cyteine

Glu Glutamate

Met Methionine

SeMet Selenomethionine

SeCyS Selenocysteine

Val Valine

v

ZUSAMMENFASSUNG

Einleitung: Selen (Se) ist ein essenzielles Spurenelement in der Ernährung von

Mensch und Tier. In der Tierernährung wird Milchkühen Selen über das Futter

supplementiert, um einerseits die Tiere vor einem Mangel zu schützen und

andererseits, um die Menge an Selen zu erhöhen, die aus dem Futter in die Milch

übergeht. Mit einer fütterungsbedingten Anreicherung von Selen in der Milch eröffnen

sich Möglichkeiten einer verbesserten Selenversorgung sowohl der Kälber als auch

die Selenversorgung der Bevölkerung über den Konsum von Milch und

Milchprodukten. Fragen der Qualität der erzeugten Milch und der daraus

hergestellten Milchprodukte sowie Fragen der Lebensmittelsicherheit insgesamt

haben dabei im Vordergrund der Betrachtungen zu stehen. Erreicht werden können

diese Ziele nur dann, wenn eine hinreichende Balance zwischen Pro- und

Antioxidantien bei der Nährstoffzufuhr gegeben ist. Selen kann durch seine

biologische Rolle bei der genetischen Kodierung von Selenocystein (SeCys),

welches als Bestandteil von Selenoproteinen vorkommt, als Antioxidations wirken.

Ziel der Arbeit war herauszufinden, ob ein erwarteter Anstieg der Selenkonzentration

in der Milch als Folge der Supplementierung der Futterrationen von Kühen mit

Selenhefe mit einer Verbesserung des Status hinsichtlich der antioxidativen

Kapazität sowie der Immunfunktionen bei den Milchkühen in der frühen Laktation

verbunden ist. Es galt die Arbeitshypothese zu testen, nach der ein verbesserter

Selenstatus der Milch auch zu einer Steigerung der Gesamtleistung der

Antioxidantien beiträgt.

Material und Methoden: 16 Holstein-Friesian-Kühe erhielten drei unterschiedliche

Futterrationen während des Zeitraums von sechs Wochen vor der Kalbung bis 15

Wochen post partum. Die tierexperimentellen Untersuchungen wurden durchgeführt

auf dem Versuchsgut des Bundesinstitutes für Risikobewertung (BfR) in Berlin. Die

Tiere der Kontrollgruppe (n = 5) erhielten dabei eine Basisration (verfüttert als Totale

Mischration, TMR) mit einem mittleren Selengehalt von ~ 0.2 mg/kg

Trockensubstanz, welches ausschließlich aus den Rationskomponenten stammte.

Die Tiere der Versuchsgruppen erhielten die Basisration, allerdings supplementiert

einerseits mit Natrium-Selenit (SeI) (n = 5) und andererseits mit Selenhefe (SeY) (n =

6), was zu mittleren Selengehalten in der TMR von 0,4 mg/kg TS bzw. 0,6 mg/kg TS

vi

führte. Die Selensupplemente wurden individuell täglich jeweils vor dem Melken in

Form von 20 bzw. 30 Gramm einer selenhaltigen Vormischung den Kühen

verabreicht. Milch- und Serumproben wurden genommen am ersten Tag nach der

Kalbung sowie in der 9., 12. und 15. Woche der Laktation. Die Selengehalte wurden

ermittelt auf Basis der Hydrid-Atomabsorptionsspektrophotometrie, die Bestimmung

der antioxidativen Kapazität (TEAC) erfolgte nach der Methode von Miller et al.

(1996), adaptiert für ein Mikroplattenphotometer. Weiterhin wurde eine Untersuchung

zum Spurenelementstatus in 11 großen Milchviehbetrieben in Sachsen durchgeführt,

um den Spurenelementstatus anhand von Futter-, Blut- und Leberbioptatproben zu

bestimmen.

Ergebnisse: Die mittleren Gehalte an Selen (± SEM) im Kolostrum der Tiere der

Kontrollgruppe bzw. derjenigen Tiere, deren Futterration mit Natriumselenit (SeI)

bzw. Selenhefe (SeY) supplementiert waren, beliefen sich auf 35,3 ± 1,03 μg/l, 39,1

± 2,56 μg/l bzw. 67,7 ± 4,11 μg/l. Die Selengehalte im Kolostrum der Tiere der

Gruppe SeY unterschieden sich dabei (P < 0.01) von denjenigen der Tiere der

beiden anderen Gruppen. Ebenso unterschieden sich aus die mittleren

Konzentrationen an Selen in der Milch (± SEM) signifikant (P < 0.01) bei den Tieren,

deren Ration mit Selenhefe supplementiert waren: 11,6 ± 1,55 μg/l (Kontrolle), 15,4 ±

3,24 μg/l (SeI) und 28,3 ± 6,84 μg/l (SeY). Die Selengehalte in der Milch der Tiere

der Kontrollgruppe und diejenigen der Kühe der Na-Selenit-Gruppe wiesen keine

statistisch gesicherten Unterschiede auf (P > 0.05), jedoch zeigte sich bei den Tieren

der Natriumselenit-Gruppe eine Erhöhung der mittleren Gehaltswerte um relativ 32%.

Die TEAC-Werte für die Tiere der SeY-Gruppe unterschieden sich in allen drei

Prüfzeiträumen (P < 0.01) sowohl von denjenigen der Tiere der Kontroll- sowie

denen der SeI-Gruppe. Insgesamt zeigten sich geringgradige Unterschiede in den

Gehaltswerten der Milch in Abhängigkeit vom Laktationsstadium. So beliefen sich die

über die Beobachtungszeiträume gemittelten Milch-TEAC-Werte (mittlere Gehalte

über den gesamten Versuchszeitraum ± SEM) auf 586 ± 0,95 μMol/l, 557 ± 0,97

μMol/l sowie 540 ± 0,64 μMol/l für die Tiere der SeY-Gruppe, der SeI-Gruppe bzw.

der Kontrolltiere. Mit Blick auf die Gehaltswerte an Selen im Serum zeigten sich

ähnlich Tendenzen wie bei den TEAC-Werten. Die Untersuchung des

Spurenelementstatus von Milchkühen zeigte eine erhebliche Variabilität bei den

Futterproben innerhalb und zwischen den Betrieben, die durch die tierbezogenen

Proben (Plasma, Leber) nicht entsprechend reflektiert wurden. Die mittleren (± SEM)

vii

Gehalte von Selen, Kupfer, Zink, Mangan und Eisen in den Leberbiopsien betrugen

0.7 (± 0.04), 134.6 (± 6.81), 18.3 (± 0.9), 7.2 (± 0.71) und 89.2 (± 6.35) mg/kg.

Schlussfolgerung: Beim Einsatz von Selenhefe in der Fütterung von Milchkühen

scheint ein Anstieg der antioxidativen Kapazität sowohl der Rindermilch als auch des

Serums hervorgerufen zu werden. Weitere Studien sind angezeigt, um die

Mechanismen aufzuklären, denen diese Effekte unterliegen.

Die Praxisstudie zeigte, dass viele Rationen für Milchkühe mehr Spurenelemente

enthalten als die gegenwärtig empfohlenen Werte. Zwischen den verschiedenen

Spurenelementen traten nach den Ergebnissen der Leberbiopsien Interaktionen auf,

deren Ursachen und Konsequenzen unter praktischen Bedingungen weiter

untersucht werden sollten.

viii

ABSTRACT Introduction: Selenium (Se) is an essential trace element for animal and human

nutrition. Dietary selenium supplementation of dairy cows is practised to protect the

animals from the risk of deficiency and to increase selenium transfer to the milk

consequently benefiting the offspring and vulnerable human populations as milk and

other dairy products make an important part of their diet. Milk quality and safety are

both important. It cannot be ensured unless a proper balance between pro and

antioxidant nutrients is maintained. Selenium, through its biological role by

genetically encoded selenocysteine (SeCyS) residue in selenoproteins, can act as an

antioxidant. The objective of this study was to find out whether the expected increase

in milk selenium levels after supplementing the dairy rations with organic selenium

yeast can affect the milk antioxidant status in the early lactating dairy cows. It was

presumed that milk total antioxidant capacity might be boosted by the enhanced milk

selenium status.

In addition to these experiments, a survey of the trace elements selenium, copper,

zinc, iron and manganese was conducted in large dairy herds of Saxonia to

determine their intake, bioavailability and interactions.

Materials and Methods: Sixteen Holstein-Friesian dairy cows were subjected to

three dietary treatment groups from 6 weeks before calving to 15 weeks of lactation

at the experimental station of the Bundesinstitut für Risikobewertung (BfR), Berlin,

Germany. The control group (n=5) was maintained exclusively on the basal total

mixed ration (TMR) containing ~ 0.2 mg/kg dietary DM selenium from the natural

sources whereas sodium selenite (SeI) group (n=5) and selenium yeast (SeY) group

(n=6) were supplemented with selenium at 0.4 mg/kg DM in the pre and 0.6 mg/kg

DM in the post partum rations respectively. Each cow received the supplement

individually in the form of 20 or 30 gram premix given before milking time. Samples

were collected at one day after calving and at 9th, 12th and 15th week of lactation.

Selenium content was analysed using the hydride generation atomic absorption

spectrometry whereas Trolox equivalent antioxidant capacity (TEAC) was measured

following the method of Miller et al. (1996) adapted for a microplate reader to

accommodate the large number of samples in duplicates.

For trace elements survey, representative TMR samples and blood and liver samples

from 11 selected farms were used to be analysed for trace element status.

ix

Results: The mean (± SEM) selenium level in colostrum for the control, SeI and SeY

groups was found to be 35.3 ± 1.03 μg/l, 39.1 ± 2.56 μg/l and 67.7 ± 4.1 μg/l

respectively in this study. Selenium yeast group was different (P < 0.01) from both

others. Average steady state milk (± SEM) selenium content was 11.6 ± 1.55, 15.4 ±

3.24 and 28.3 ± 6.84 μg/l for control, SeI and SeY groups respectively with SeY

group differing (P < 0.01) from other groups. Control and SeI groups were not

different (P > 0.05); however a relative increase of 32% was noted in SeI group. It

has been noted that TEAC values for the SeY group were significantly different (P <

0.01) from that of control and SeI groups at all time points. However, negligible

differences have been observed between different time points in all groups. Milk

TEAC values of (mean of all time points ± SEM) were 586 ± 0.95 μMol/l, 557 ± 0.97

μMol/l and 540 ± 0.64 μMol/l for the SeY, SeI and control groups respectively. Similar

trends in serum selenium and TEAC values have been noted. The investigation of

the trace element concentrations in the total mixed rations of dairy cows indicated a

huge variability within and between the farms that were not clearly reflected by the

plasma and liver samples taken from the animals.

The mean (± SEM) concentrations of selenium, copper, zinc, manganese and iron in

the fresh liver biopsy samples from Saxonian dairy herds were 0.7 (± 0.04), 134.6 (±

6.81), 18.3 (± 0.9), 7.2 (± 0.71) and 89.2 (± 6.35) mg/kg respectively.

Conclusion: This study reveals some sort of selenium-related increase in the total

antioxidant capacity of bovine milk and serum. This can have implications for the

health of the animals and public health concerns over milk safety. Further studies will

help delineate the actual underlying mechanisms. Survey findings revealed that

generally there is a trend of supplementing the dairy rations with trace elements

above the requirements. Positive and negative interactions among the trace

elements have been observed and will need further studies to explain effects under

practical conditions.

1

1. INTRODUCTION Efficient livestock and poultry production and the maintenance of normal health in

animals require that essential nutrients be provided in appropriate amounts and in

forms that are biologically utilizable. Deficiencies of certain nutrients occur in diets

consisting of common feed ingredients and this has led to the common practice

around the globe of supplementing the diets of farm animals with essential nutrients.

Degree of the bioavailability of the nutrients does not only influence the dietary

requirement but also the tolerance for a nutrient. Advances in the nutritional

technologies have resulted in the development of innovative products to be used as

animal feed supplements. These products must be designed to deliver the

incremental nutrients in a safe and economical way in the food chain. Among various

products used as animal feed supplements, amino acids, macro and micro minerals

and enzymes are most important and popular. The trace element selenium (Se) has

attracted substantial research efforts during the current and the last decade owing to

its special place in the animal and human nutrition. Its essentiality and the toxicity are

within narrow margins. Essentiality of this nutrient is based on its major role in the

antioxidant defence system of the living cells.

Apart from being naturally found as sodium selenate (Na2SeO4) and sodium selenite

(Na2SeO3), selenium can be incorporated biologically in proteins containing

methionine. Plants and yeast exposed to selenium salts accumulate the trace mineral

in the form of selenomethionine (Se-Met). Sodium selenite and selenium enriched

yeast are in common use as sources of selenium in farm animals. Although

substantial amount of work has been carried out in the field of selenium nutrition of

dairy cows, gaps still exist in the knowledge regarding comparative efficacy of

supplementation from various sources. Moreover, some work in this regard has been

done in Germany. It has been shown in several studies that dietary selenium yeast

significantly increases selenium concentrations in blood, milk and other tissues as

compared to inorganic selenium sources. Phenomenon of non-specific pooling of

selenomethionine from selenium yeast into tissue proteins instead of methionine is

accounted for this increase. However, Juniper et al. (2006), after conducting an

experiment with selenium supplementation in the range of 0.27-0.4 mg/kg DM with

2

selenium yeast, reported that only 25-33% of total milk selenium increase could be

attributed to selenomethionine and there are other selenoproteins in milk which might

play a role as an antioxidant. Hence, the present study investigates the effect of

selenium supplementation from sodium selenite and selenium yeast on the selenium

status and Trolox equivalent antioxidant capacity (TEAC) in pregnant and lactating

cows and their calves. It is hypothesized that increased selenium status in the

supplemented cows’ serum and milk will be reflected in the form of heightened

antioxidant status. No such attempt has been made previously to get information

regarding the effect of selenium from sodium selenite and selenium yeast on the total

antioxidant capacity in dairy cows. This study provides basic information on the topic

in addition to generate the data on selenium and revolves around the following

objectives:

Investigations into selenium and antioxidant status on various time points of

physiological importance during the periparturient and lactation stage

The assessment of selenium transfer into milk, risk assessment depending on the

dietary level and source of selenium

Selenium transfer to calves and its impact on their health and well-being

Studies into the intake, bioavailability and interactions among essential trace

elements in large dairy herds under practical conditions

3

2. REVIEW OF LITERATURE This chapter is based on the review article “The Role of Dietary Selenium in the

Bovine Mammary Gland Health and Immune Function” by Salman et al. (2009).

2.1 Selenium: From Toxicity to Essentiality

Selenium (Se, atomic number 34 and atomic weight 78.96) is placed in 4th period and

16th group of metalloids and non-metal chemical elements of the periodic table. Many

of its chemical properties (outer valence electronic configuration, atomic size, bond

energy, ionization potential and electronegativity) are similar to that of sulphur.

Selenium occurs in oxidation states –II (selenide), 0 (elemental selenium), +IV

(selenite) and +VI (selenate) forms. In isolated form, it is found like grey-black

metallic cluster.

Discovered by Jöns Jacob Berzelius in 1817, the semi-metal selenium was named

after the Greek Goddess of the moon, Selene (McKenzie et al. 1998). Dietary

importance of selenium dates back in history when it was first reported to cause the

toxic symptoms in the members of the caravan of the great adventurer, Marco Polo.

Livestock disorder, commonly referred as alkali disease or blind stagger, was found

endemic in areas with selenium rich soils. Similarly, symptoms of chronic selenium

intoxication, depression and fatigue, and loss of hair and nails, were noticed in

human beings living geographic in regions with high soil selenium before it was

known to be the causative agent. That is why early scientists showed interest in

selenium because of its toxic effects. However, the approach towards selenium

research in life sciences began to change as early as 1916 when selenium was

detected in normal human tissue samples. It was suggested “it may have a position

in the organism which will without doubt be of the utmost significance in the study of

life processes” (Gassmann 1916). The earliest evidence that selenium is involved in

the immune function was found in 1957 with the observation that dogs injected with 75Se incorporated the isotope into a leukocyte protein (now known to be the

cytoplasmic glutathione peroxidase cGSHPx) (Schwarz and Foltz 1957). In sheep

and humans, selenium is concentrated in tissues involved in the immune response

such as spleen, liver and lymph nodes (Spallholz 1990). The question how this trace

4

element exerts its biochemical role was solved when it was discovered in 1973 to be

the essential component of GSHPx and the cellular antioxidant defence system

(Rotruck et al. 1973). The subsequent discoveries in rats about the fact that two

thirds of the dietary selenium are not bound to this enzyme but are part of other

compounds (Behne and Wolters 1983) led to the assumption that other

selenoproteins may exist. Thus far 55 selenoproteins, including glutathione

peroxidases (1-6), thioredoxin reductases (1-3) and iodothyronine deiodinase

families of selenoenzymes have been reported. Consequently, dietary selenium

deficiency has been known to cause various ailments in a number of animal species

and humans. Keshan and Kashin-Beck diseases in humans, muscular dystrophy in

sheep and cattle and exudative diathesis in poultry are notable among selenium

deficiency disorders. This voyage of selenium from toxicity to essentiality is still in

progress with revelation of new discoveries and facts about selenium and its related

compounds and their role in diverse physiological functions of the body. The narrow

margin of safety (average dietary intake for selenium and the tolerable upper intake

level for both sexes has been reported by National Research Council (2001) as 113-

220 μg and 400 μg/day respectively for adult humans) is sufficient to stress its

importance in the diets.

2.2 Selenium and Mechanism of Oxidative Stress

Oxygen is the prerequisite of life and ultimate source of energy for its sustainability.

Animals, plants and many microorganisms rely on oxygen for efficient energy

production. In doing so, free radicals capable of initiating further chain reactions are

generated. These free radicals are capable of damaging the biologically relevant

molecules such as DNA, proteins, lipids and carbohydrates. Superoxide (O2-) is the

main free radical produced in biological systems during normal respiration in

mitochondria and by autooxidation reactions at 37°C. It is notable that superoxide, by

itself, is not extremely dangerous and does not rapidly cross the lipid membrane

bilayer. However, it is a precursor of other more powerful free radicals collectively

known as reactive oxygen species (ROS) and reactive nitrogen species (RNS). An

imbalance in the production and accumulation of these highly reactive oxygen

species (ROS) - activated derivatives of molecular oxygen, including singlet oxygen,

O2-, H2O2, hydroxyl radical, hypohalous acids and peroxynitrites - may lead to the

most inevitable of the biological problems, the oxidative stress, because it derives

5

from the least-specific type of reaction: univalent electron transfer which can occur if

the oxygen species come across with the redox cofactors at a lower potential than

themselves . Reactions of this type (Figure 1) are responsible both for the formation

of ROS and for their subsequent inactivation of various biomolecules. It has been

experimentally manifested in the E. coli devoid of cytoplasmic superoxide dismutase

(SOD) that these strains grew well anaerobically but exhibited a variety of aerobic

growth defects that derived from endogenous O2-. Similarly, E. coli

catalase/peroxidase mutants were poisoned by micromolar levels of H2O2 that

accumulated inside the cell (Park et al. 2005). Both sets of mutants exhibited

catabolic and biosynthetic defects that stem from the inactivation of a family of

dehydratases.

Figure 1 The standard concentration of oxygen was regarded as 1M. Abbreviations: H2O2, hydrogen peroxide; O2

-, superoxide (Imlay 2008)

The other best-understood mechanisms of oxidative injury involve the oxidation of

inactivation of exposed enzymic iron-sulphur clusters and the production of hydroxyl

radicals within proteins and on the surface of DNA (Imlay 2008). Superoxides can

also participate in the production of powerful radical ions by donating an electron and

thereby reducing. It is speculated that basic biochemistry of the oxidative damage is

likely shared by most cells, and most contemporary organisms have inherited from

their ancestors a common set of strategies by which to defend themselves.

Although much remains to be understood about how cellular defences against the

oxidative stress work, through its natural homeostatic balance the animal body must

be able to keep free radicals in control. Defensive tactics revealed thus far include

various free radical scavenger enzymes and isozymes for example superoxide

dismutase, catalases, peroxidases and repair mechanisms. Inability or loss of

oxidant-resistance strategies can be manifested in terms of many disease conditions

in man and animals.

Figure 1 The redox states of oxygen with standard reduction potential (volts).

6

The transition period and early lactation in dairy cows is critically important for health,

production and profitability (Drackley 1999). Dairy cows vigorous physiological

activities during periparturient period concerning the rapid differentiation of secretary

parenchyma, intense mammary gland growth and the onset of copious milk synthesis

and secretion are accompanied by high energy demand and increased oxygen

requirement . This increased oxygen demand can result in the augmented production

of ROS, which are potential source of the cells and tissues injury, commonly referred

as the oxidative stress leading to a high susceptibility of dairy cows to a variety of

infections and metabolic disorders during the transition period. Vulnerability of the

transition period in cattle is marked by reproductive problems and prevalence of

mastitis. This can be ascribed to findings that various components of the host

defence mechanisms, particularly the immune cells, are depressed during this

period. It has been reported that functional capabilities of mammary macrophages

decrease during the periparturient period and this alteration has been linked with an

increased incidence of mastitis. Presence of neutrophils at the site is inversely

correlated with the risk of mammary infections. In vitro efficacy of neutrophils

obtained from selenium-deficient mice, rats and cattle in killing ingested microbes is

significantly reduced as compared to that from selenium-sufficient animals. It is

because of the reduced activity of the antioxidant enzyme Glutathione peroxidase

(GSHPx), responsible to protect neutrophils to be damaged by their own superoxide-

derived radicals, in selenium-deficient animals as selenium is an integral component

of the enzyme. Supplementing the dairy rations with vitamin E and selenium has

become a widely accepted practice throughout the world to address the issue of

prooxidants and antioxidant balance. As being an essential component of the

GSHPx, selenium is able not only to convert toxic hydrogen peroxides to water but

also the lipid hydroperoxides to non reactive compounds participating in the

antioxidant defence system of the body at initial and secondary levels of blocking the

chain of reactions .

Selenium performs its biological role through the genetically encoded selenocysteine

residue (SeCys) of selenoproteins. Selenium can affect three broad areas of cellular

functions: antioxidant activities, thyroid hormone metabolism, and the regulation of

redox-active protein activity. Out of 30-50 known selenoproteins (Köhrle 2000) at

least 12 have been relatively well characterized as having wide-ranging implications

7

for immune function, malignancy and viral pathogenesis. The best-known

selenoenzyme with respect to dairy cattle nutrition is glutathione peroxidase

(GSHPx). Indeed, it is an essential component of the cellular antioxidant defence

mechanism, which removes potentially damaging lipid hydro-peroxides and hydrogen

peroxides and protects the immune cells from oxidative stress induced damage. A

recent report describes that thioredoxin reductase (TrxR) may be an important

antioxidant defence mechanism in peripheral blood mononuclear cells (PBMC) that is

compromised during the periparturient period. Indeed the most of the functional

capabilities of selenoproteins are related to their crucial role in regulating the ROS

and redox status in nearly all tissues. However, some effects on the regulation of

arachidonate metabolism in peripheral blood lymphocytes resulting in the partial

reversal of proliferation have also been reported. New insight in the role of free

radicals as signalling molecules and understanding the role of nutrients in gene

expression have created new demands for further research related to the biological

roles of selenium.

2.3 Metabolism of Selenium in Mammals

It is interesting to note that selenium is unique in its metabolism compared with

typical essential trace elements such as copper and zinc. As with other dietary

nutrients, selenium from organic and inorganic dietary sources has to be metabolized

by the ruminal microorganisms before being absorbed by separate mechanisms in

the small intestine of ruminants. Not much is known about selenium metabolism in

the rumen. In sheep, ruminal absorption of 75Se has been reported to be only 34%

probably because of the conversion of dietary selenium to insoluble forms such as

elemental selenium and selenide (Spears 2003). More recently, it has been

demonstrated that inorganic selenium has a lower ruminal microbial uptake than

organic selenium sources in dairy cows (Mainville et al. 2009). In the small intestine,

amino acid derivatives of selenium (selenomethionine and selenocysteine), mainly

found in the organic selenium sources such as selenium yeast, use the same carriers

as their sulphur analogues methionine and cysteine (Glass et al. 1993), whereas

selenate uses a sodium sulphate cotransporter for its absorption, which is driven by

the activity of Na+/K+-ATPase at the basolateral enterocyte membrane (Mehta et al.

2004). In the lumen of the small intestine, selenite partially reacts with glutathione or

other thiols to selenotrisulfides, which are presumably taken up into the enterocytes

8

by amino acid transporters. Another part of selenite diffuses through the apical

membrane and reacts with thiols in cytosol of enterocytes. Subsequently, selenium

compounds are liberated in the blood stream at the basolateral enterocytes

membrane and distributed to various peripheral tissues. The exact transport

mechanism of various selenium compounds is not yet fully understood.

Selenomethionine associates with hemoglobulin while selenate and the remaining

free selenite were found to be transported by � and �-globulins (Beilstein and

Whanger 1986b, a). Ionic selenium forms of selenite and selenate follow bicarbonate

and phosphate, respectively, in their transport in the body because of similarity in

their ionic forms (Suzuki 2005). In fact selenite ions are readily taken up by red blood

cells (RBCs) through band three protein without being excreted into urine (Suzuki et

al. 1998) while selenate ions are not taken up by RBCs but directly taken up by

hepatocytes through transport system of phosphate and partly excreted directly into

urine (Kobayashi et al. 2001). Selenite taken up by RBCs is readily reduced to

selenide and then effluxed into the blood stream in the presence of albumin and

transferred to liver in the form bound to albumin (Shiobara and Suzuki 1998). It can

be concluded that selenide of selenite and selenate origin are taken up differently by

the liver and utilized for the synthesis of selenoproteins. A surplus of inorganic

selenium is stored in peripheral organs as “acid labile selenium”. This selenium

fraction consists of selenium bound unspecifically to proteins presumably via the

formation of selenium-sulphur bonds (Diplock et al. 1973; Ganther and Kraus 1984).

The main excretion products of selenium detected in urine are the methylated

metabolites monomethylselenol (MMS) and trimethylselenonium (TMS). Methylated

selenium metabolites are formed from selenium reduced to the oxidation state –II as

well as from selenium stored unspecifically in proteins as selenomethionine and from

acid labile selenium (Hassoun et al. 1995). Selenium exhalation as dimethylselenide

only takes place when selenium is ingested in toxic doses. The metabolism and the

fate of dietary selenium has been summarised demographically in the following

representations (Figure 2 and Figure 3).

9

Figure 2 Selenium incorporation in proteins (Suzuki 2005)

Figure 3 Selenium metabolism in mammals (Suzuki 2005)

2.4 Selenium Nutrition of Dairy Cows

The nutritional status of the animal is related to its overall health and its capacity to

combat disease. The nutritionally modulated improvement of the immune system

should culminate in increased resistance to disease. Research on micronutrients and

their immunoregulatory role regarding udder health and bovine mastitis has focused

mainly on selenium, vitamin A, vitamin E, �-carotene, copper and zinc. Among these,

10

selenium has been the most characterized trace element affecting bovine mammary

gland health through its role in cell function.

Having been recognized as a dietary essential, selenium is being routinely

supplemented in the rations of farm animals. In the United States, 0.1 mg selenium

/kg dry matter (DM) is recommended for ruminant rations to correct symptoms of a

selenium deficiency. However, owing to the beneficial effects of the additional

selenium supplementation, the recommendation was increased to a level of 0.3

mg/kg DM (National Research Council 2001). The German Society for Nutritional

Physiology (GfE) has recommended that selenium intake levels for dairy cattle

should range from 0.2 mg/kg DM (GfE 2001) whereas the recommendations by the

British authorities are 0.1 mg/kg DM (MAFF 1983). Supplementation of this nutrient

to dairy animals can be one of the best options, not only to protect the animal from

disease threats, but also to raise the selenium level in milk and subsequently transfer

this essential element to the human population, many of whom are marginal deficient

in selenium.

2.5 Biomarkers for Selenium Status

The scientific controversy regarding the identification of the best biomarker for

selenium status assessment is still unresolved. In dairy cows, several approaches

have been followed to assess the status of the herd or the individual animal. These

approaches include the direct estimation of selenium in whole blood, serum or

plasma, milk and others tissues of interest; and indirect measures such as the intra-

and extra-cellular activity of the selenium containing enzyme, glutathione peroxidase

(GSHPx) in whole blood, serum or plasma. A number of studies have shown that

serum selenium or GSHPx activity represents the short-term selenium status, while

parameters for the whole blood or erythrocytes reflect the long-term selenium status.

Stowe and Herdt (1992) determined the reference range of serum selenium level of

70-100 ng/ml. This value has been described as an adequate level. Earlier reports

(Maus et al. 1980; Detoledo and Perry 1985) suggested that an adequate selenium

level in blood serum should be in the range of 40-120 ng/ml. Variations in these

findings may be the result of dietary concentration and nutritional management

practices. Gerloff (1992), on review of the data from various research groups,

considered the value of 70-100 ng/ml for serum selenium as a consensus of opinion

11

regarding the adequacy of selenium, particularly when the dietary source is inorganic

selenate or selenite.

With the discovery, that glutathione peroxidase (GSHPx) has selenocysteine as its

essential component; the activity of this enzyme has been regarded as the pertinent

parameter for the assessment of selenium status. Although numerous studies have

associated the activity of GSHPx with the selenium status of the animal because of a

linear response of GSHPx activity with selenium supplementation, GSHPx activity as

the parameter of selenium status assessment has been criticized (Stowe and Herdt

1992). Inconsistency of units used in expressing the enzyme activities, difficulty in

ensuring the proper storage conditions of samples, enzyme concentrations that reach

a plateau while serum selenium concentrations continue to rise and delayed

response to supplementation and different cellular and extra cellular forms are all

points which need to be taken into account when considering GSHPx activity as a

criterion for selenium status of the animal. On the other hand, the relationship

between GSHPx and health is better explained than between plasma selenium

concentration and health. Awadeh et al. (1998a) showed that only one-third of total

selenium intake is incorporated into GSHPx, and that GSHPx activity is largely

confined to the erythrocytes.

Milk selenium concentrations can potentially be used as a simple parameter for the

selenium status assessment of dairy herds. In a study conducted with large dairy

herds over several seasons, a sigmoid relationship with an adjusted R2 value of .92

(P < 0.0001) was observed between the bulk tank milk selenium and mean serum

selenium values (Wichtel et al. 2004). A plateau effect was noted in serum selenium

concentrations when milk concentrations exceeded 20 μg/l. Tentative reference

values for bulk tank milk selenium have been generated based on the relationship

observed. Milk selenium concentrations less than 9.6 ng/ml are considered to

indicate a deficiency, whilst a value of 21.8 ng selenium/ml appears to represent an

adequate selenium supply. The value 15.7 ng/ml is the median between the marginal

range of the low and high categories. However, it is notable that the source of the

selenium has not been kept considered while making the bulk tank milk selenium as

an accurate measure of the herd selenium status. Many studies have reported that

milk selenium concentrations were significantly higher when diets were

12

supplemented with selenium yeast as compared to sodium selenite at the same level

(Ortman and Pehrson 1999; Muniz-Naveiro et al. 2005; Juniper et al. 2006). Positive

correlations, irrespective of the source of selenium supplementation, of 0.59, 0.64

and 0.68 have been observed between the cows’ milk and their calves erythrocytes

GSHPx activity, whole blood, and plasma selenium concentrations, respectively

(Pehrson et al. 1999). A cautious estimate of the herd selenium status can be made

by bulk tank milk selenium concentrations, keeping the source of selenium

supplementation in mind.

The source and dietary level of the nutrient are important in determining the

nutritional status of the animal. Different supplements of selenium are categorised

based on organic and inorganic forms. Sodium selenite and sodium selenate are

common inorganic forms whereas the organic form of selenium is produced from the

yeast Saccharomyces cerevisiae, with almost 90% of the total selenium represented

by selenomethionine (Muniz-Naveiro et al. 2005). As far as the bioavailability of

selenium from organic versus inorganic sources is concerned, whole blood selenium

concentration, GSHPx activity and milk selenium concentration in dairy cattle

increase more efficiently after dietary selenium supplementation using organic

sources compared to inorganic ones (Malbe et al. 1995; Awadeh et al. 1998b;

Knowles et al. 1999; Ortman and Pehrson 1999; Gunter et al. 2003). However,

selenium yeast and selenite follow a similar pattern of distribution among serum

proteins (Awadeh et al. 1998b). Cattle fed selenium yeast have a higher percentage

of selenium in whole blood (average 20%), milk (average 90%) and increased activity

of GSHPx (16%) compared to cattle fed inorganic selenium (Weiss 2005).

Previously, Knowles et al. (1999) had reported no difference in the blood GSHPx

activity between cows fed selenite and those fed a selenium yeast compound,

provided the cows consumed 4 mg/day of supplemental selenium (approximately 0.2

ppm). However, when cows were fed 2 mg/day, the GSHPx activity was 50% higher

than when selenium yeast was used as source of dietary selenium. Comparative

increases in milk and blood selenium levels after supplementing the diet of cows with

a selenium yeast source have been largely attributed to non-specific incorporation of

selenomethionine from the diet into the tissue proteins (Weiss 2005). However,

Juniper et al. (2006), after conducting a study with selenium supplementation in the

range of 0.27-to 0.4 ppm from a selenium-containing yeast source, reported that only

13

25-33% of total milk selenium increase could be attributed to selenomethionine and

that there are other selenoproteins in milk, which might play a role as an antioxidant.

Interactions between the selenium status of dairy cows and the udder defence

system have been explored. Parameters of milk somatic cells and microbial counts,

incidence and duration of clinical mastitis cases in dairy herds, and controlled

experiments with or without the experimental challenge of pathogenic microbes, have

been the prime focus in this area. With the advent of selenium yeast products on the

market, research is now focussing on safety and comparative efficacies. Based on

the information cited above, it can be inferred that the selenium status of the animal

is directly correlated with dietary level and source, and organic selenium sources

tend to be comparatively more efficient in maintaining the selenium status of the

animal than are inorganic sources.

2.6 Somatic Cell Count and Selenium Status

The somatic cell count (SCC) of milk is used as a benchmark parameter to estimate

udder health and consequently milk quality. Cell concentration of the milk varies

widely as a function of the lactation cycle. In healthy udder conditions, very few

leukocytes should migrate into milk during full lactation. At cessation of milking, the

SCC might increase owing to the intense physiological changes occurring in the

udder. Milk from a healthy bovine udder should contain very few somatic cells (<

20,000/ml), and whenever the SCC rises above 20,000/ml, there has been

histological evidence of inflammation in the udder (Schalm et al. 1971). Rainard and

Riollet (2006) reported that the SCC in most uninfected and uninflamed quarters is

considerably less than 100,000/ml, with a low portion of neutrophils, which can

increase up to 40% near the drying off period. Somatic cell concentrations increase

to reach 2-5 ×106/ml during the first 7-10 days of the dry period. They then remain

stabilized in the range of 1-3 ×106/ml. After parturition, the SCC decreases to 105/ml

in the first 7-10 days after calving.

Higher SCC values in milk reflect a diseased udder making the milk less valuable. It

is evident (Table 2) that milk SCC is negatively correlated to the selenium status of

the animal. It was reported that the cow’s udder is more prone to infection if GSHPx

activity in the blood is below 3.3 μkat/g of haemoglobin (Malbe et al. 2003). Lack of

14

GSHPx activity causes oxidative damage to soft tissue, thus making the udder more

vulnerable to mastitis pathogens. Consequently, infiltration of neutrophils in the udder

tissue will cause the SCC to rise to higher levels. The effective role of neutrophils in

combating the microbial threat is also dependent on GSHPx activity. Enhanced

viability and vitality of neutrophils in response to optimum GSHPx activity could be a

plausible explanation for the low SCC in the milk of cows having improved selenium

status and consequent enhanced GSHPx activity.

Few studies failed to find a correlation (Grace et al. 1997; Wichtel et al. 2004)

between the selenium status of cows and disease susceptibility. This has been

attributed to the fact that the data involved the results of surveys conducted with

herds having different management practices. Marginal bulk tank milk selenium

levels of (0.018 μg/ml), and corresponding marginal serum selenium levels, could

have been the reason why Wichtel et al. (2004) did not find any substantial

relationships between bulk tank milk selenium levels and the general parameters

used to assess udder health.

2.7 Mastitis Susceptibility and Selenium Status

Low selenium status is linked to increased susceptibility of dairy cows to

intramammary infections (Table 2). Marked reduction (up to 60%) in infected

mammary gland quarters has been observed in dairy cows after selenium

supplementation for a period of 8 weeks at 0.2 ppm dietary level (Malbe et al. 1995;

Ali-Vehmas et al. 1997). Duration of clinical mastitis was reduced by 46% in cows

supplemented with selenium and by 62% in cows supplemented with selenium and

vitamin E (Smith et al. 1984).

Supplementation with selenium and/or vitamin E at levels far above those required

for growth and normal physiological function can result in the improvement of various

components of the immune system and general animal health (Surai 2006). This is

particularly important for cows infected with pathogens. In an experiment described

by Hemingway (1999), 14 of 36 cows receiving intramammary antibiotic infusions at

drying off needed extra treatment in the subsequent lactation whereas only 5 of 36

cows which received additionally 4 mg selenium at drying off needed such treatment.

Udder health benefits have been attributed to antibacterial activities against S.

15

aureus in milk whey protein (Ali-Vehmas et al. 1997; Malbe et al. 2006). The

underlying mechanism of this antibacterial activity is not well understood. However, it

was proposed that impaired microbial growth rate in the whey fraction exhibiting high

GSHPx activity may account for the results. The absence of both glutathione and

GSHPx in bovine milk has been reported (Stagsted 2006). Therefore, further

generation of more reactive radical oxygen species by phagocytes or the presence of

other selenoproteins in milk may account for the results obtained. It can be

concluded that selenium may affect mastitis susceptibility of the mammary gland by

improving the phagocyte recruitment to the infected quarters, increasing their vitality

and inducing unspecified antibacterial activity in milk whey against various

pathogens.

2.8 Mammary Gland Immune System – Interactions with Selenium

The immune response is characterized by heterogeneity of reactive cells and their

products, having specificity for the response and memory following subsequent

antigen exposures. The bovine mammary gland produces colostrum which is rich in

antibodies that can protect the newborn from infectious agents (Sordillo et al. 1997).

The bovine mammary gland is itself protected by a variety of defence mechanisms,

which can be separated into two distinct categories: innate immunity and adaptive

immunity, each having sensing and effectors arms (Rainard and Riollet 2006). The

innate and acquired immune systems interact closely in an attempt to provide

protection against pathogens (Sordillo et al. 1997; Burvenich et al. 2003). The

acquired immune response uses many innate immune effector mechanisms to

eliminate microorganisms and its action frequently increases innate antimicrobial

activity (Oviedo-Boyso et al. 2007). The efficacy of the adaptive immune response

rests in its specificity, memory of the immune cells and also, to some extent, on the

immune stimulus, which is augmented by repeated exposure to the antigen. On the

other hand, innate immunity is non-antigen-specific, exists prior to the encounter with

the pathogens, and is related to the processes of acute and chronic inflammation and

sepsis (Finlay and Hancock 2004).

16

2.8.1 Physical Barriers The first lines of defence against foreign molecules and invading pathogenic

microorganisms are the natural physical barriers of the body. Mastitis can occur

when bacteria gain entrance into the mammary gland via the teat canal. The teat end

contains sphincter muscles that maintain tight closure between milkings and hinder

bacterial penetration. Increased patency of these muscles is directly related to an

increased incidence of mastitis (Murphy and Stuart 1953; Myllys et al. 1994). The

teat canal is lined with keratin, which is crucial to the maintenance of the barrier

function of the teat and removal of the keratin correlates with increased susceptibility

to bacterial invasion and colonization (Capuco et al. 1994; Sordillo and Streicher

2002). Teat keratin is a waxy material derived from stratified squamous epithelium

that traps invading bacteria and exhibits bactericidal properties (Hibbitt et al. 1969;

Craven and Williams 1985). Esterified and non-esterified fatty acids (myristic,

palmitoleic and linoleic) function as bacteriostatic agents, and are associated with

keratin of the teat canal (Miller et al. 1992). More recently, it has been noted that

certain cationic proteins associated with keratin can bind to pathogenic

microorganisms, thus increasing their susceptibility to osmolarity changes leading to

the lyses and death of the invading pathogens (Paulrud 2005). Because of the

efficacy of the teat canal barrier, the intra-mammary lumen is an aseptic chamber to

which the aseptic character of normal milk can be attributed. Thus, the teat canal is

an important barrier against intra-mammary infections.

There may be a role for selenium in teat canal keratin function as it has been found

that in mammalian spermatozoa phospholipid hydroperoxide glutathione peroxidase,

a selenoprotein, is functionally associated with the cross linking of the structural

elements of the cytoskeleton via the oxidation of high sulphur keratin-associated

proteins (Maiorino et al. 2005a; Maiorino et al. 2005b). There is no direct evidence of

the association of selenium with the bovine mammary gland teat canal.

2.8.2 Cellular Factors

Bacteria and other pathogens, upon entry into the body tissues, are only able to

cause disease by overcoming the body’s natural cellular defence mechanism.

Different types of cells in combating the pathogens play a pivotal role. Cellular factors

17

of the bovine mammary gland immune system come from two main types: the

mammary epithelial cells (MECs) and the immune cells comprising macrophages,

neutrophils, Natural Killer (NK) and dendritic cells. Collectively these constitute the

somatic cells of the milk.

Mammary epithelial cells (MEC) were previously considered the major cell type in

milk (Schalm et al. 1971). However, a later study confirmed that MECs are rarely

found in the milk and the major cell type of the tissue and secretion of the bovine

mammary gland is the macrophages (McDonald and Anderson 1981). The presence

of sub- and intra-epithelial leukocytes, and the repertoire and distribution of sensor

receptors on MECs makes the immune system of the mammary gland peculiar,

resembling the urinary tract system and differing from the intestine (Rainard and

Riollet 2006). Mammary epithelial cells express mRNA for TLR 2, 4 and 9 and �-

defensin 5, thus contributing positively towards the sensing of pathogens

(Goldammer et al. 2004). Adhesion of bacteria and the interaction of bacterial toxins

with the epithelial cells has been reported to induce the synthesis of tumour necrosis

factor alpha (TNF-�), interleukin-6 (IL-6) and IL-8 (Rainard 2003).

Phagocyte Responses

Much of the uptake of foreign antigens is performed by macrophages, neutrophils

and natural killer cells in the mammary gland. During the defence of the mammary

gland against bacterial infection, tissue and milk macrophages recognise the

invading pathogen and initiate the inflammatory response by releasing pro-

inflammatory cytokines (TNF�- and IL-1�), that induce neutrophils recruitment to the

mammary gland (Bannerman et al. 2004).

Macrophages are the major cell type in milk, secretions of the involuted udder, and

mammary tissue (Jensen and Eberhart 1981; Mcdonald and Anderson 1981).

Although macrophages can ingest common mastitis pathogens, they are less active

phagocytes than are milk neutrophils. Furthermore, both milk cell types are less

efficient than their blood counterparts (Mullan et al. 1985). In addition to phagocytic

activity, macrophages also play a role in antigen presentation (Politis et al. 1992) and

are responsible for the removal of neutrophils following the elimination of bacterial

pathogens. The functional capabilities of mammary macrophages decrease markedly

18

during periparturient periods and this alteration has been linked to an increased

mastitis incidence (Waller 2000; Sordillo and Streicher 2002). Apart from the stress

associated with parturition and the start of lactation, the underlying mechanism of the

periparturient immunosuppression is still unclear.

Ndiweni and Finch (1995) worked with bovine mammary gland macrophages

obtained from cows fed a selenium adequate diet. They investigated the effect of

various doses of vitamin E, sodium selenite and combination of both on cellular

functions in vitro. Sodium selenite supplementation in vitro from 1 nM-10 μM to S.

aureus-stimulated macrophages enhanced the production of chemotactic factors

significantly (P < 0.003). Similar effects were recorded with vitamin E

supplementation in the range from 5 ng/ml to 50 μg/ml. There were no synergistic

effects of both nutrients. Concentrations of selenium above 0.1 mM depressed

chemotaxin production. It was suggested that the stimulatory effect of selenium might

be attributed to its role as cofactor of LTB4 synthase or hydrase, as peritoneal

macrophages from rats fed selenium-deficient diets are not able to produce a

respiratory burst reaction and as a result, their antimicrobial function is compromised

(Parnham et al. 1983).

Neutrophil numbers in normal milk from healthy bovine mammary gland are too low

for efficient phagocytosis (Leijh et al. 1979). Pro-inflammatory cytokines released by

macrophages and MECs activate the expression of cellular adhesion molecules by

endothelial cells that cause the binding and subsequent migration of blood

neutrophils from blood to the site of infection, or in the milk where they are further

localised. Following bacterial entry into the mammary gland, neutrophils are the first

cells that are recruited into the milk and represent the predominant cell type.

Neutrophils recruitment from the circulation to the site of infection is essential in the

defence of the mammary gland against invading bacteria. The promptness of the

recruitment and the number of recruited neutrophils, which vary in intensity according

to pathogen type and the cow, determines the outcome of the infection.

Neutrophil concentrations increase rapidly between 3-12 h post-challenge and can

reach more than 107/ml in milk following E. coli infusion in the mammary gland,

whereas in the case of a S. aureus challenge, the recruitment is delayed (between

19

24-48 h and remains below 106/ml (Riollet et al. 2000; Rainard and Riollet 2006).

Recruited neutrophils at the site of infection phagocytose bacteria and produce

reactive oxygen species, low molecular weight antibacterial peptides, and defensins,

which eliminate a wide variety of pathogens (Mehrzad et al. 2002; Paape et al. 2002;

Sordillo and Streicher 2002; Paape et al. 2003). The increase in the concentration of

milk neutrophils is in fact the origin of high SCC during mastitis and this is the reason

why their presence is inversely correlated with the risk of intramammary infections

(Burton and Erskine 2003).

The most important and widely investigated association between selenium and the

immune function in dairy cows is the effect of this micronutrient on neutrophils

function. Neutrophils perform their microbe killing function by producing super-oxide

derived radicals. This type of process is a balance between sufficient radical

production for microbial killing and the system that protects the neutrophils

themselves from these radicals. This balance is attributed to the cytosolic glutathione

peroxidase activity within the neutrophils, which is impaired in selenium deficiency,

which permits neutrophils to be self-destroyed. The earliest evidence regarding the

effect of selenium on neutrophils function was reported by Boyne and Arthur (1979).

In that study, it was noted that the ability of neutrophils to phagocytise Candida

albicans cells was not different (P < 0.05) between selenium-deficient and selenium-

supplemented calves receiving 0.1 mg of dietary selenium/day. However, the number

of neutrophils with the ability to kill phagocytosed C. albicans cells was about three

times less for selenium-deficient animals having undetectable levels of blood GSHPx

activity. On the other hand, both phagocytosis (P < 0.05) and killing (P < 0.01) of S.

aureus by blood PMN leukocytes were higher (P < 0.05) when the dairy cows

received between 10-17 mg selenium/day, along with an additional 350-1000 mg

vitamin E/day for a period of 16 days (Gyang et al. 1984). However, phagocytosis by

neutrophils from cattle supplemented with selenite or selenate at low levels (2

mg/day or 0.2 mg/kg DM, respectively) was not different from that of neutrophils from

unsupplemented cows.

Direct and indirect measures of bacterial killing were higher (P < 0.05) in neutrophils

isolated from selenium-supplemented cattle as compared to those from

unsupplemented cows (Grasso et al. 1990; Hogan et al. 1990). In a survey

20

conducted by Cebra et al. (2003) higher blood selenium levels (> 300 ng/ml) were

associated with enhanced neutrophils adhesion and intracellular kill by the

neutrophils obtained from post parturient cows. With PMN cells isolated from the

blood of selenium-adequate cows, it was found that in vitro supplementation of

selenium (10 μM) had greater stimulatory effect (129%) on their random migration

than did vitamin E (71%) and, at the highest concentration of selenite used (1 mM),

random migration of PMN was inhibited (Ndiweni and Finch 1996). On the other

hand, vitamin E enhanced phagocytosis of S. aureus to a greater extent than did

sodium selenite after a 2 h incubation period (Ali-Vehmas et al. 1997). Both nutrients

were not significantly different in their ability to stimulate PMN cells to produce

superoxide. Enhanced recruitment of neutrophils at the site of infection in selenium-

supplemented cows has also been reported previously (Ali-Vehmas et al. 1997).

Organic and inorganic sources of selenium at 0.3 mg/kg DM intake have been

compared for their effect on the function of neutrophils obtained from the blood of

lactating cows (Weiss and Hogan 2005). There were no significant differences

regarding either the ability of neutrophils to phagocytise bacteria or the percentage of

E. coli that were killed, although there was a slight increase in the percentage kill for

the selenium yeast group. These observations agree with those of Malbe et al.

(1995) regarding the effect of selenium source on bovine neutrophils’ phagocytosis of

S. aureus. A plausible explanation for this effect might be the non-specific pooling of

selenomethionine from organic selenium sources into tissue proteins instead of

methionine and the presence of 0.2% sulphur in the diets. However, it is difficult to

interpret such data, as a negative control was not included. More recently, Mukherjee

(2008) has reported an improvement (P < 0.05) in phagocytosis of S. aureus by milk

neutrophils obtained from mastitic riverine buffaloes that had been injected with a

selenium/vitamin E preparation containing sodium selenite and had been treated with

enrofloxacin.

Lymphocyte Responses

Long-term cellular specific immunity is a function of both antigen-presenting cells and

lymphocytes, which are the only cells of the immune system that recognize antigens

by membrane receptors specific to invading pathogens. If the invading pathogens

survive the activities of macrophages and neutrophils, T and B lymphocytes and

21

monocytes become the predominant cell type. Leitner et al. (2003) observed that

lymphocytes were the most common infiltrating cell type within the two-layer

epithelium lining the teat cistern; monocytes and macrophages were present in lower

number. Nevertheless, neutrophils remain most important in chronic mastitis

(Rainard and Riollet 2006). T lymphocytes are classified into two main groups: T��

and T��. T�� include CD4+ (helpers) and CD8+ (suppressors) cells. In healthy

mammary glands CD8+ lymphocytes are the prevailing type, whereas in mastitis

infected mammary glands CD4+ cells are predominantly activated by the formation of

a molecular complex between the major histocompatibility complex class II (MHC II)

and antigens presented by B lymphocytes and macrophages (Park et al. 2004).

Through their ability to secrete certain cytokines, CD4+ cells help B lymphocytes to

proliferate and secrete antibodies. CD4+ cells are mainly found in the inter-alveolar

tissue of the mammary gland whereas CD8+ cells surround the alveoli (Leitner et al.

2003).

In contrast with the milk, cells obtained from blood exhibit a higher ratio of CD4+ to

CD8+ cells; however, the functional significance of this elevated frequency has not

been clearly established. CD8+ cells may be either cytotoxic or suppressor type. Post

partum they are mainly of the cytotoxic type, whereas during mid and late lactation

they are of the suppressor type (Sordillo et al. 1997). Cytotoxic T cells recognise and

eliminate altered self cells via antigen presentation in conjunction with MHC I

molecules. They act as the scavengers of old and damaged secretory cells and their

secretions are related to the susceptibility of the bovine mammary gland to infections

(Oviedo-Boyso et al. 2007). Although T�� cells are not well characterized, they are

associated with the epithelial surface where they destroy damaged epithelial cells

(Yamaguchi et al. 1999).

Natural Killer cells, B cells and dendritic cells are also part of the bovine mammary

gland immune system. Natural Killer (NK) cells are large granular lymphocytes that

have cytotoxic activity independent of MHC, through antibody-dependent cell

mediated cytotoxicity. In contrast to neutrophils and macrophages, they are critical to

the removal of intracellular pathogens. Bovine NK-like cells (CD2+ CD3- T

Lymphocytes), express bactericidal activity against S. aureus upon stimulation with

IL-2 in a non-specific manner (Sordillo et al. 2005). These cells destroy both gram

22

positive and gram-negative bacteria and are fundamental to the prevention of bovine

mammary gland infections (Sordillo and Streicher 2002). The primary role of B

lymphocytes is to produce antibodies against invading pathogens. In doing so, they

utilize their cell surface receptors to recognize specific pathogens and process the

antigens. Processed antigens are thus presented to T helper cells, which secrete

cytokine IL-2 that, in turn, induces the proliferation and differentiation of B

lymphocytes into either plasma cells that produce antibodies or memory cells. Not

much is known about the density and role of dendritic cells in the bovine mammary

gland immune system. Normally they are associated with antigen presentation.

It has been suggested that selenium and vitamin E deficiencies affect T lymphocytes

to a greater extent than B lymphocytes (Larsen et al. 1988). This was suggested to

be the result of higher levels of polyunsaturated fatty acids in T lymphocytes and

associated with higher membrane fluidity. Selenium and vitamin E deficiencies may

affect both the maturation of specific lymphocyte subpopulations and proliferative

capabilities of peripheral lymphocytes (Surai 2006). In an experiment with dairy cows

fed either basal diet (~ 0.05 mg selenium/kg DM) or a diet supplemented with sodium

selenite (~ 0.20 mg selenium/kg DM), it was noted that Con A stimulated lymphocyte

proliferation was significantly higher in the selenium-supplemented group (Cao et al.

1992). Similar findings have been reported when bovine peripheral blood

lymphocytes were supplemented with sodium selenite in vitro from 1 nM to 10 μM

concentrations (Ndiweni and Finch 1995).

Selenium supplementation or deficiency in mice altered the kinetics of IL-2 receptor

expression (Roy et al. 1994). Supplementation in vitro or in vivo resulted in an earlier

expression of high affinity IL-2 receptors, whereas selenium deficiency resulted in a

delayed expression of receptors. This may explain the stimulatory role of selenium in

the enhanced T cell function. In healthy aged humans, selenium supplementation

(400 μg/day, for 6 months) enhanced NK cell cytotoxicity over pre-treatment levels by

58% (Wood et al. 2000). There is no information on the effect of selenium on NK cell

and dendritic cell function in dairy cows. It is interesting to note that enhanced

immune cell function resulted from selenium supplementation levels, which are

higher than normally recommended.

23

2.8.3 Soluble Factors

Soluble factors of the bovine mammary gland immune system are made up of

various proteins that include complement proteins, cytokines and immunoglobulin.

Each class performs its physiologically defined function with a high level of

specificity.

The bovine complement system is a collection of proteins that is present in serum

and milk, and has an important role in the defence of the mammary gland.

Complement proteins are predominantly produced by hepatocytes, though they are

also produced by monocytes and macrophages in different tissues. In the presence

of antibodies, they lyse invading pathogens. Complement component C3b binds the

antibody bacteria complex for efficient phagocytosis by neutrophils and macrophages

(Paape et al. 2003) whereas C5a stimulates the recruitment of neutrophils, which

augments their phagocytic and bactericidal activities (Rainard and Poutrel 2000).

Cytokines are produced by both immune and non-immune cells and are essential in

almost all aspects of host defence. They regulate the activities of cells involved in the

immune function. A variety of cytokines such as interleukins (IL) -1�, -2, -6, -8, -12,

colony stimulating factor (CSF), interferon gamma (IFN-�) and TNF-� have been

detected in healthy and infected bovine mammary glands (Sordillo and Streicher

2002; Alluwaimi 2004). TNF-� is the main cytokine produced by macrophages,

neutrophils and epithelial cells during the early stage of infection and participate in

the neutrophil chemotactic activity (Persson et al. 2003). CD4+ and CD8+

lymphocytes and NK cells in response to mitogenic and antigenic stimuli produce

IFN-�. Interferon-� functions in activating the acquired immune response and

phagocytic activity of neutrophils and is important in viral infections (Shtrichman and

Samuel 2001). Monocytes, macrophages, and epithelial cells produce IL-1�. During

the inflammatory response, IL-1� regulates the expression of adhesion molecules

and neutrophils chemotaxis in E. coli infections (Yamanaka et al. 2000). IL-2,

produced by CD4+ lymphocytes, regulates the acquired immune response by

stimulating the growth and differentiation of B lymphocytes and the activation of NK

and T cells. Alterations in IL-2 production cause a decrease in the mammary gland

24

immune response capacity, which facilitates mastitis (Sordillo et al. 1991; Sordillo

and Streicher 2002).

Immunoglobulins (Ig) are synthesized by plasma cells that are differentiated from B

lymphocytes upon activation by IL-2. In milk, immunoglobulin either are synthesized

locally or originate from blood (Sordillo and Nickerson 1988). The role of antibodies in

the natural defence mechanisms of the udder is to opsonise bacterial pathogens,

thereby aiding the neutrophils and macrophages in phagocytosis.

Four classes of Ig are known to influence mammary gland defence against bacteria

causing mastitis: IgG1, IgG2, IgA and IgM. Each of these classes differs in

physicochemical and biological properties (Gershwin et al. 1995). The concentration

of each immunoglobulin in the mammary secretion varies with the stage of lactation,

increasing during dry periods and approaching peak concentrations during

colostrogenesis (Sordillo and Nickerson 1988). The largest part of the opsonic

antibodies in adult serum and milk of cows is IgM (Williams and Hill 1982; Hill et al.

1983). The presence of IgM in cows, without a previous history of mastitis, suggests

that they are mainly auto-antibodies directed against self-antigens and are poly-

reactive in nature (Rainard and Riollet 2006). In this regard, the cow is not different

from humans or rodents, who also have these types of antibodies in their blood (Saini

et al. 1999).

Non-specific proteins such as lactoferrin, lysozyme, transferrin, xanthine oxidase and

the lactoperoxidase system, exhibit bacteriostatic and bactericidal activities against

common mastitis pathogens. In normal function, various components of the innate

and adaptive immune system are coordinated to provide protection to animals

against invasion by pathogens.

Improved selenium status of animals results in enhanced immunoglobulin titre in

colostrum from cows receiving a high dose of selenium, administered by

intramuscular injection pre-partum (Pavlata et al. 2004). Earlier studies reported

lower (P < 0.05) concentrations of IgG and IgM in plasma and colostrum of beef cows

and calves fed a free-choice salt/mineral mixture containing 20 ppm selenium as

sodium selenite, compared to the cows and calves fed a salt mixture containing 60

25

ppm selenium in the form of selenium yeast compound, or 120 ppm sodium selenite.

The source of selenium affects only the IgM concentration of plasma with higher

concentrations (P < 0.05) when cows are fed a selenium yeast supplement (Awadeh

et al. 1998b).

It was recommended that consideration should be given to the concentrations of T3

(thyroid hormone) and IgG whilst determining the nutritional requirement of cattle for

selenium. Swecker et al. (1989) also confirmed higher concentrations of colostral Ig

G in beef cows fed higher selenium levels in free choice mineral mixtures. Enhanced

proliferation of B lymphocytes in cell cultures containing 100 ng/ml selenium

suggests a mechanism for the increased IgM production (Stabel et al. 1991).

Contradictory observations of a no effect (P < 0.05) of selenium on the

immunoglobulin have also been reported (Lacetera et al. 1996; Leyan et al. 2004). It

is noteworthy that positive effects have been observed only when higher selenium

doses were used. Depression in several leukocyte function parameters, including the

forced antibody response, was noted in pre-parturient beef cows consuming 6 ppm

or 12 ppm selenium as sodium selenite from their diets (Yaeger et al. 1998).

Studies on the interactions of selenium with the immune system of the mammary

gland, general udder health and mastitis susceptibility are summarized in Table 1

and Table 2. There is little data on the effect of selenium on cytokines and other

soluble factors in dairy cows available.

26

Tabl

e 1

Dai

ry c

attle

imm

une

resp

onse

s as

affe

cted

by

sele

nium

R

efer

ence

St

udy

Type

Se

leni

um

Supp

lem

enta

tion/

Con

cent

ratio

nSe

leni

umSo

urce

Im

mun

eR

espo

nse

Stud

ied

Mat

rix

Obs

erva

tions

Wei

ss a

nd H

ogan

(2

005)

C

ontro

l E

xper

imen

t w

ith E

.Col

i ch

alle

nge

0.3

mg/

kg D

M in

die

t S

elen

ium

ye

ast a

nd

Sod

ium

S

elen

ite

Neu

troph

il fu

nctio

n B

lood

N

eith

er p

hago

cyto

sis

nor

perc

enta

ge k

ill w

as

sign

ifica

ntly

affe

cted

by

sele

nium

sou

rce

Pav

lata

et a

l. (2

004)

C

ontro

l E

xper

imen

t 44

-88

mg

IM in

ject

ion

Sod

ium

S

elen

ite

Imm

unog

lobu

lin

Col

ostru

m

Sig

nific

ant i

ncre

ase

(P <

0.0

5)

in tu

rbid

ity u

nits

was

obs

erve

d in

sup

plem

ente

d gr

oup

Ceb

ra e

t al.

(200

3)

Sur

vey

>

300

ng/m

l in

bloo

d S

odiu

m

Sel

enite

N

eutro

phil

func

tion

Blo

od

Incr

ease

d ad

hesi

on o

f ne

utro

phils

and

incr

ease

d in

trace

llula

r kill

alo

ng w

ith

high

er m

ilk p

rodu

ctio

n in

cow

s w

ith h

igh

sele

nium

sta

tus

Pan

ousi

s et

al.

(200

1)

Con

trol

Exp

erim

ent

IM in

ject

ion

of 0

.1 m

g/kg

bod

y w

eigh

t S

odiu

m

Sel

enite

S

peci

fic

antib

odie

s ag

ains

t E. C

oli

Ser

um

Ser

um c

once

ntra

tion

of

spec

ific

antib

odie

s ag

ains

t E.

coli

incr

ease

d (P

< 0

.05)

in

supp

lem

ente

d co

ws

at d

ay 6

3

Cao

et a

l. (1

992)

C

ontro

l E

xper

imen

t 0.

05-0

.2 m

g/kg

DM

S

odiu

m

Sel

enite

Ly

mph

ocyt

es

Blo

od

Sig

nific

antly

enh

ance

d (P

<

0.05

) lym

phoc

ytes

pro

lifer

atio

n w

ith C

on A

was

obs

erve

d in

th

e ce

lls fr

om s

elen

ium

su

pple

men

ted

cow

s du

ring

48-

96 h

ours

G

rass

o et

al.

(199

0)

Con

trol

Exp

erim

ent

with

E .C

oli

chal

leng

e

2 m

g/da

y in

die

t (90

day

s)

Sod

ium

S

elen

ite

Neu

troph

ils

func

tion

Milk

P

hago

cyto

sis

rem

aine

d un

affe

cted

but

sig

nific

ant

incr

ease

(P<

0.05

) in

killi

ng o

f in

gest

ed b

acte

ria w

as

obse

rved

in s

uppl

emen

ted

cow

s

27

Tabl

e 2

Bov

ine

udde

r hea

lth a

nd m

astit

is s

usce

ptib

ility

as

affe

cted

by

sele

nium

5

Ref

eren

ce

Stud

y Ty

pe

Sele

nium

Supp

lem

enta

tion

/Con

cent

ratio

n

Sele

nium

Sour

ce

Para

met

ers

Stud

ied

Obs

erva

tions

Muk

erje

e R

. (20

08)

Con

trol

Exp

erim

ent

1.5

mg/

day

in th

e fo

rm o

f in

tram

uscu

lar

inje

ctio

n (5

day

s)

Sod

ium

S

elen

ite

SC

C

GS

HP

x S

CC

dec

reas

ed s

igni

fican

tly(P

< 0.

05) f

rom

296

1x10

3to

630

x103

in

buffa

loes

scr

eene

d po

sitiv

e fo

r int

ra m

amm

ary

infe

ctio

ns w

here

as G

SH

Px

activ

ity in

crea

sed

sign

ifica

ntly

Mal

be e

t al.

(200

6)

Con

trol

Exp

erim

ent

4 m

g/da

y in

die

t (8

wee

ks)

Se-

yeas

t M

ilk p

rote

ins

antib

acte

rial

activ

ity

agai

nst S

.au

reus

G

SH

Px

Sel

eniu

m s

uppl

emen

ted

cow

s ex

hibi

ted

prof

ound

ant

ibac

teria

l act

ivity

in

milk

whe

y fra

ctio

ns w

hen

the

activ

ity o

f blo

od G

SH

Px

incr

ease

d si

gnifi

cant

ly fr

om <

1.0

2 μk

at/g

Hb

to >

4 μ

kat/g

Hb

Kom

mis

rud

et a

l. (2

005)

S

urve

y 20

-230

μg/

l in

bloo

d U

nspe

cifie

d S

CC

M

astit

is

Ret

aine

d P

lace

nta

Sig

nific

antly

low

(P =

0.0

3) b

ulk

milk

SC

C (1

37x1

03 /ml)

was

obs

erve

d in

he

rds

with

hig

h bl

ood

sele

nium

leve

l as

com

pare

d to

155

x103 /m

l in

herd

s w

ith lo

w b

lood

sel

eniu

m le

vel.

Red

uced

inci

denc

es o

f dis

ease

trea

tmen

t re

gard

ing

mas

titis

and

reta

ined

pla

cent

a w

ere

obse

rved

in a

nim

als

with

hi

gh b

lood

sel

eniu

m le

vels

Ju

kola

et a

l. (1

996)

S

urve

y

191

μg/l

in b

lood

U

nspe

cifie

d S

CC

In

cide

nce

of

clin

ical

m

astit

is

A 1

7.7%

and

70.

6% d

ecre

ase

in in

fect

ions

cau

sed

by S

. aur

eus

and

Cor

yneb

acte

rium

spe

cies

resp

ectiv

ely

was

foun

d to

be

asso

ciat

ed w

ith

high

blo

od s

elen

ium

leve

l

Wic

htel

et a

l. (1

994)

C

ontro

l E

xper

imen

t 6-

12 m

g/da

y (w

hole

lact

atio

n)

Sod

ium

S

elen

ite

SC

C

Sig

nific

ant d

ecre

ase

(P <

0.0

2) in

SC

C fr

om th

e le

vel o

f 235

x103 /m

l to

112x

103 /m

l in

diffe

rent

her

ds w

ith s

elen

ium

sup

plem

enta

tion

was

ob

serv

ed

Mad

dox

et a

l. (1

991)

C

ontro

l E

xper

imen

t w

ith E

. col

i ch

alle

nge

0.05

-0.3

5 m

g/kg

D

M

Sod

ium

S

elen

ite

Milk

bac

teria

l co

unt

Milk

bac

teria

l cou

nt w

as s

igni

fican

tly h

ighe

r(P

< 0

.05)

in s

elen

ium

-def

icie

nt

grou

p an

d th

is g

roup

requ

ired

ther

apeu

tic tr

eatm

ent w

here

as

supp

lem

ente

d gr

oup

reco

vere

d w

ithou

t the

rape

utic

inte

rven

tion

Wei

ss e

t al.

(199

0)

Sur

vey

70-9

0 μg

/l (h

erd

mea

n pl

asm

a)

Uns

peci

fied

SC

C

Inci

denc

e of

m

astit

is

Sig

nific

ant (

P <

0.0

5) n

egat

ive

corr

elat

ions

(-0.

84, -

0.68

) wer

e ob

serv

ed

betw

een

herd

mea

n pl

asm

a se

leni

um c

once

ntra

tion

and

SC

C in

the

rang

e of

724

-744

x103 /m

l and

mas

titis

inci

denc

e du

ring

the

who

le la

ctat

ion

28

2.9 Concluding Remarks

Survey findings and controlled studies with or without experimental challenge

indicate a role for selenium in the immune function and improvement in bovine

mammary gland health. Although selenium status has been noted to increase

markedly as a result of the supplementation with selenium yeast as compared to

inorganic sources, whether this increase is completely translated in terms of health

benefits to the animal is not clear. Most recent findings have confirmed that selenium

levels higher than those considered adequate can potentially enhance the natural

defence mechanisms of the bovine mammary gland at maximum, especially the

humoral responses.

There are limited studies on the clinical aspects of the health of the bovine mammary

gland, as affected by organic versus inorganic selenium sources, or a combination of

the two sources of selenium. Moreover, there are many gaps in our knowledge of the

interactions of selenium with the immune function of the bovine mammary gland.

Neutrophilic function has been the major point of focus of the research. Other

aspects of the immune response, notably, the activity of Natural Killer (NK) cells as

affected by selenium supplementation in combating both gram-positive and gram-

negative mastitis pathogens, has not been studied. Furthermore, certain cytokines

and mammary epithelial cells and lymphocyte proliferation response have great

implications for mammary gland health and mastitis control. More work is required to

delineate these interactions.

29

3. MATERIALS AND METHODS

3.1 Feeds and Animals

The experiment was performed with 16 pluriparous Holstein-Friesian cows

maintained at the research station of German Federal Institute for Risk Assessment

(BfR) in Marienfelde, Berlin (50°, 24.6´, N; 13°, 22.1`). The research station is

facilitated with the modern individual feeding chambers and milking parlour. The

individual cow data were recorded using leg-band transponders fitted on the cows.

All the experimental cows were in between their 1st and 3rd lactation and calved

during June 2008 to February 2009. At drying off, cows were blocked based on parity

and expected calving date into three groups (5, 5 and 6 cows) in a way to have

minimum variation regarding the parity between different groups and then randomly

allotted to receive additional supplementation. Each cow received either selenium

supplement or placebo individually in addition to the basal diet (Table 3) containing

0.15-0.20 mg selenium/kg DM presuming a dry matter intake of 10 Kg daily and

offered in the form of total mixed ration during the experimental period. Organic and

inorganic selenium supplements were prepared by mixing the ground corn with Sel-

Plex-1000 (Batch No. 71658-2, CNCM-I 3060) and sodium selenite (Na2SeO3)

supplements obtained from the local feed company to give the final selenium content

of 200 mg/kg in the product. All the cows were given a three months adaptation

period with the basal diet before the start of the experiment. Each cow was fed 20

gram of either supplement or placebo at the time of morning milking during the pre

partum period and 30 g during the post partum experimental period. Supplementation

corresponded to an additional intake of 4 and 6 mg selenium/day during the

prepartum and postpartum experimental phases respectively.

30

Table 3 Composition of total mixed ration (TMR) fed as basal diet during the feeding trial

Ingredients %TMR Selenium (µg/kg DM) ±SEM n

Maize silage 75.5 12.2 222653

Hay 4.4 20.6 Straw 4.4 27.0 Beet pulp 4.4 153.2 16.66 Soybean meal 2.2 227.4 15 Rapeseed meal 4.4 87.6 9 Vitamin-mineral mix1 4.4 1680.5 110.4 5Milk concentrate2 177.7 10.22 9

Nutrient composition

Dry matter (%) 49.5 0.88 6Crude protein (%DM) 10.7 0.005 6Crude fat (%DM) 2.7 0.005 6Crude ash (%DM) 5.3 0.015 6Crude fiber (% DM) 17 0.15 6Neutral detergent fiber (%DM) 53.1 4.05 4Acid detergent fiber (%DM) 17.3 0.277 4

1 Contains 5.5% Ca, 1.5% P, 2.5% Mg, and 4.2 % Na, 460000 IU vitamin A, 33500 IU vitamin D3, 500 IU vitamin E and 490 mg CuSO4.5H2O per kg; 3.6 MJ NEL/kg 2 Contains 0.78% Ca, 0.5% P, 0.3% Na, and 10000 IU vitamin A, 800 IU vitamin D3, 90 IU vitamin E and 13 mg CuSO4.5H2O per kg; 7.0 MJ NEL/kg; offered extra as 1 kg for every 3 kg increase in milk production during the lactation

Table 4 Mineral composition (DM basis) of total mixed ration (TMR) fed as basal diet during the feeding trial (n=3)

Minerals Mean ±SEM Calcium (g/kg) 5.6 0.06 Phosphorus (g/kg) 3.4 0.02 Sodium (g/kg) 1.6 Magnesium (g/kg) 2.2 Potassium (g/kg) 10.7 0.07 Manganese (mg/kg) 87.0 0.73 Copper (mg/kg) 19.0 1.09 Cobalt (mg/kg) 0.7 0.06 Zinc (mg/kg) 136.0 0.54 Iron (mg/kg) 398.7 2.58 Selenium (mg/kg) 0.18 0.01

31

3.2 Sampling

All the procedures regarding the management and sampling from the animals were

approved by the Landesamt für Gesundheit und Soziales (LAGeSo). Cows were

sampled 6 and 3 weeks before anticipated calving, within 12 hours after the calving,

and 1 and 12 weeks after calving for blood samples. Colostrum and milk samples

were collected at day 1 after calving and 1, 9, 12, and 15 weeks after calving.

Aliquots of milk samples were frozen at -80°C for subsequent analysis.

3.3 Chemicals and Instruments

Selenium standard solution (1000 mg selenium/l) and hydrochloric, nitric and

perchloric acids were purchased from Merck (Darmstadt, Germany). Skim milk

powder (Standard reference material, NIST 8435) was obtained from LGC Standards

(Wesel, Germany) whereas, 2, 2´-azinobis (3-ethylbenzothiazoline 6-sulfontate)

(ABTS), Trolox standard antioxidant (6 Hydroxy, 2, 5, 7, 8-tetramethylchroman 2-

carboylic acid) and activated manganese oxide were purchased from Sigma-Aldrich

(Steinheim, Germany). The atomic absorption spectrometer (Vario 6 equipped with

H52 hydride system and auto sampler) was made by Analytik Jena AG (Jena,

Germany) whereas microplate reader (Sunrise TC) was from Tecan (Salzburg,

Austria).

3.4 Estimation of Selenium

Total selenium in feeds, supplements and milk was estimated by the hydride

generation atomic absorption spectrometry (HG-AAS). Samples were digested using

a programmable electrically heated digestion block (Tecon, TZP-500). The digestion

process was carried out in a mixture of nitric and perchloric acids by using the quartz

digestion tubes. In the end stage of digestion process 6M hydrochloric acid was

added in the tubes to reduce selenium (VI) to selenium (IV) for hydride generation in

the system. The samples were diluted before measurement to a final volume of 40

ml. The method was standardised using whole milk powder (Standard reference

material, NIST 8435). The analyses of the milk standard reference material resulted

in 121.8 ± 6.62 μg/kg (mean ± SD, n=15) as compared to the reference range value

of 131.0 ± 14 μg/kg. Reference standards were used for every twenty analyses. Ultra

pure deionised water of 18.2 M� cm (4 ppb TOC) obtained from Milli-Q apparatus

was used for making dilutions and washing.

32

3.5 Estimation of Antioxidant Activity

Total antioxidant activity was measured using the Trolox Equivalent Antioxidant

Capacity (TEAC) according to the method of Miller et al. (1996). However, the

method was modified keeping in view the changes suggested by Wang et al. (2004)

regarding the endpoint measurement and adapted to carry out large number of

samples in the standard conditions using the microplate plate reader. The TEAC

assay was originally based on the suppression of the absorbance of the radical

cations of 2, 2´-azinobis (3-ethylbenzothiazoline 6-sulfontate) (ABTS) by antioxidants

in the test sample when ABTS (Figure 4) incubates with peroxidase (metmyoglobin)

and H2O2. The modified procedure requires the production of long living radical cation

(ABTS•+) by the action of ABTS and activated manganese oxide. Briefly, pure ABTS

was dissolved in 5 mM PBS buffer with pH 7.4 to have a final solution of 5 mM ABTS.

The solution was filtered through Wattmann filter paper across the activated

manganese oxide while keeping it under light protection for 12-16 hours for efficient

radical generation. The filtrate was finally passed through 0.2 μm syringe filter (VWR-

cellulose acetate) and kept under light protection. Standard calibration curve was

generated with the average of two values corresponding to blank, 50, 100, 150, 200

and 250 μM/l Trolox solution prepared from 97% Trolox standard antioxidant (6-

hydroxy-2, 5, 7, 8-tetramethylchroman 2-carboylic acid) for the each run. The

absorbance was recorded at 620 nm after the inhibition period of 20 minutes in the

96-well micro plate containing 190 μl of ABTS•+ and 10 μl pre-diluted sample (milk).

Figure 4 Chemical structure of ABTS molecule

3.6 Statistical Analysis

Data obtained was analysed statistically using SPSS 15 (Chicago, USA). Dunett’s

test of Post Hoc comparisons was performed for significance testing of the means of

various groups in a multivariate ANOVA (Field 2005). This test was applied because

it tests the significance of means of treatment groups in comparison with that of a

control. Level of statistical significance was set as 0.05 during the data analysis.

Correlations and regression equations were computed using the same software.

33

4. Results

4.1 Colostrum and Milk Selenium Status

The mean (± SEM) selenium level in colostrum for the control, SeI (Sodium Selenite)

and SeY (selenium yeast) groups was 35.3 ± 1.03 μg/l, 39.1 ± 2.56 μg/l and 67.7 ±

4.11 μg/l respectively in this study. Statistical analysis has revealed that mean

colostrum selenium content of the SeY group is different (P = 0.032) from that of the

SeI and control group animals (P = 0.018). Furthermore, no difference (P = 0.754)

has been observed between the SeI and control groups regarding colostrum

selenium levels. In control group, colostrum selenium content ranged from 20.6 –

60.4 μg/l, whereas for SeI and SeY groups this range was found to be as 25.9 - 58.0

and 39.9 – 106.7 μg/l respectively. The large variation in colostrum selenium content

might be attributed to the genetic factors and to some extent to the health problems

as the intake of the supplement and the placebo was not largely different in all

groups.

In milk, a decrease of 60, 42 and 35 percent has been observed in selenium content

after one week of calving for the control, SeI and SeY groups respectively. It can be

assumed from the results of the present research that milk selenium content has a

declining trend still the steady state is obtained after about 12 weeks of milking. The

average steady state milk (± SEM) selenium content for the control, SeI and SeY

groups has been noticed as 11.6 ± 1.55, 15.4 ± 3.24 and 28.3 ± 6.84 μg/l,

respectively. Statistical analysis of the data revealed that control and SeI groups milk

selenium content at first week after calving was nearly different (P = 0.072) and after

ninth weeks of calving it differed significantly (P < 0.05). No difference (P > 0.05)

could be found between control and SeI groups after 12 and 15 weeks of the

experimental period. It could also be observed that SeY group exhibited more

variations in terms of standard deviations as compared to both others. The results

have been shown graphically in the following figure 5.

34

Figure 5 Colostrum and milk selenium concentrations in various treatment groups.

Each graphical symbol represents the mean ± SEM of respective treatment groups. SeY group differs (P < 0.05) from other groups at all time points. SeI and control cows are not different (P > 0.05)

4.2 Milk Trolox Equivalent Antioxidant Capacity (TEAC)

Milk Trolox equivalent antioxidant capacity (TEAC) values for various treatment

groups in the study have been graphically represented in Figure 6. The results are

the mean of duplicate values measured for each sample on the specific time point

regarding the day of lactation. Each group’s samples have been measured on a

separate 96 well microplate. Separate calibration curves for Trolox concentrations

ranging from 0-250 μM/l generated for each microplate. From the regression

equation and the trend line it was evident that standard calibration curves exhibited

good linearity (R2 = .998 - .999) and were within the comparable absorption range. It

has been noted that TEAC values for the SeY group are significantly different (P <

0.001) from that of control and SeI groups at all time points and SeI group differed (P

< 0.001) from that of control in the same manner. However, negligible differences

have been observed between different time points in all groups. Milk TEAC values of

(mean of all time points ± standard error) have been observed as 586 ± 0.95 μMol/l,

35

557 ± 0.97μMol/l and 540 ± 0.64 μMol/l for the SeY, SeI and control groups

respectively.

Figure 6 Milk TEAC values at various lactation stages in different groups.

All groups differ (P < 0.01) from each other. There is no difference within the groups (P > 0.05)

4.3 Milk Production

Milk production data has been presented in the Table 5. Milk yield was recorded

digitally at the milking parlour during the milking process of the individual cows. Milk

samples from each cow were taken fortnightly during the experimental period for

subsequent analysis of the milk nutrients. No differences among the control and

treatment groups have been observed regarding the milk and nutrient yield. Weiss

and Hogan (2005) have reported the same previously. It can be assumed from the

milk and nutrients yield data that experimental cows were normal producers.

36

Table 5 Milk and nutrients yield in different treatment groups during the feeding trial

Table 6 demonstrates that treatment groups did not differ much regarding their reproductive

and udder health. However, it can be noted that SeI group was less concerned whereas in both

other groups’ number of affected animals remained three during the experimental period.

Seeing the total number of treatments, it can be observed that udder health disorders are not

much different whereas SeY group suffered more (9) as compared to SeI (2) and control (5)

with regard to reproductive health disorders.

Table 6 Health status of experimental cows during feeding trial

Cows No. of times treated Udder Health Reproductive Health Control 1 + 4 + 1 2 + 1 + 2 SeI 1 + 4 1 + 1 SeY 2 + 3 + 1 3 + 1 + 5

Each digit represents an animal and number of treatments it received during the feeding trial.

4.4 Serum Selenium in Cows

Figure 7 exhibits the profile of serum selenium concentration in various treatment

groups on different sampling time points starting from 6 weeks before anticipated

calving until 12th weeks after calving. Experimental cows were not different (P > 0.05)

before supplementation started at 6th week before expected calving. Trend lines for

the control and treatment groups follow different pattern. Control group cows serum

selenium content declines 3 weeks before calving and at the calving from 47.9 ± 5.94

μg/l (Mean ± SEM) to 38.5 ± 4.67 μg/l and 30.6 ± 9.88 μg/l, respectively, after which it

appears to plateau around 35.0 ± 6.56 μg/l. Trend in both the supplemented groups

resembles showing a relative decrease of 8% and 16 % at calving for SeY and SeI

37

groups reactively. This decrease at calving in control group was found as 30%. It has

been noted that both groups differ significantly (P < 0.05) from that of control except

that SeI groups is not different (P = 0.141) at calving from both others. However, it is

indicated that in spite of substantial relative increase, 8-34% at various time points in

the serum selenium level of selenium yeast supplemented cows, SeI and SeY groups

are not different (P > 0.05) in raising the selenium content of serum in dairy cows.

However, P values were noted to be decreasing as 0.476, 0.385, 0.178 and 0.08 at

three weeks before calving, at calving, one week after calving and 12 weeks after

calving respectively.

Figure 7 Serum selenium concentrations of dams in different groups during various physiological stages.

SeY and SeI groups differ (P < 0.05) from Control cows at all time points except at calving when only SeY group is different

4.5 Serum Selenium Level in Calves

Serum selenium content in calves from various treatment groups and at different time

points has been shown in the following Figure 8. Some significant differences in

groups and regarding time points have been observed. The control group was

significantly different (P < 0.05) from SeY group at both the time point (within 12 hrs

after calving and one week after calving). SeY group was also significantly different

38

(P < 0.05) from SeI groups at both time points, however, SeI group calves were only

different from control groups calves at calving in their serum selenium contents.

Serum selenium content at calving for the control, SeI and SeY groups’ calves has

been noted to be (Mean ± SEM) 23.5 ± 2.01, 29.1 ± 3.5, and 38.1 ± 0.96 μg/l

respectively. Whereas one week after calving respective contents increased for

different groups as 28.4 ± 2.03, 37.7 ± 1.23 and 47.7 ± 1.05 μg/l.

Figure 8 Serum selenium concentrations of calves borne to dams in different treatment groups.

Different superscripts within groups and time points denote significant differences (P < 0.05)

4.6 Body Mass of Calves

Body mass of dams and calves measured at various time points have been

presented in Table 7. None of the values was different (P > 0.05). However, calves

live body weight after one week of age was little higher (52 kg as compared to 50 kg)

in SeY group. This can be attributed to the fact that 4 out of 5 calves in SeY group

were male. Dams live body mass decreased at the end of the experimental period

due to calves’ birth.

39

Table 7 Body mass (kg) of cows and calves in various treatment groups

Groups Cows (kg) Calves (kg)

In the start At the end birth After 1 week

Control 654 ± 11 552 ± 47 43 ± 2 50 ± 2 P > 0.05

SeI 703 ± 19 633 ± 23 43 ± 1 50 ± 21 P > 0.05

SeY 672 ± 42 644 ± 5 46 ± 1 52 ± 2 P > 0.05

1 n = 4 ; 2 n = 6 ; *All values are mean ± standard error rounded to nearest kg (n = 5). No difference among groups at a single time point (P > 0.05)

Figure 9 Differences in body mass of calves at various time points

No statistical difference among various groups could be noted.

4.7 Serum TEAC in Cows

Serum TEAC was measured in cows’ samples obtained within 12 hours of calving,

and one and twelve weeks after calving. The results of serum TEAC in dams have

been presented in Figure 10. Although TEAC values were found within a narrow

range (566 – 577 μMol/l), statistical analysis revealed (P < 0.001) differences among

the treatment groups. SeY group exhibited the highest values compared to other

groups in the study. Slightly increased TEAC values at calving time suggest some

sort of homeostatic mechanism to counteract the oxidative stress in this period. It has

also been noted that SeY group is less different (P = 0.056) from other groups in

40

decrease in its TEAC values at 12 weeks of sampling time. No difference (P > 0.05)

has been observed in TEAC at calving and one week after calving in all groups. The

mean serum TEAC values (± SEM) for all time points in control, SeI and SeY groups

have been found to be 566 ±1.39, 570 ± 0.61 and 577 ± 0.50 μMol/l respectively.

Figure 10 Serum TEAC values in cows of various treatment groups.

Groups differ with each other (** = P < 0.01)

4.8 Serum TEAC in Calves

Serum TEAC values were measured in calves within 12 hrs after birth and one

week after calving. The results have been shown graphically in Figure 11. It is

notable that TEAC values in calves’ serum followed a decreasing trend as in

dams regarding time after calving. However, in calves, TEAC values one week

after calving were lower (P < 0.01) as compared to the values in prior samples

obtained within 12 hrs after calving. This is also in contrast to their dams

serum TEAC values, which were not found different (P > 0.05) one week after

calving. The average TEAC values ( Mean ± SEM) for the control, SeI and

SeY groups within 12 hrs after calving have been found to be 567 ± 0.25, 571

± 0.12 and 578 ± 0.5 μMol/l respectively. Whereas, one week after calving

41

TEAC values decreased in their respective groups as 566 ± 0.25, 570 ± 0.16

and 577 ± 0.15 μMol/l.

Figure 11 Serum TEAC in calves of various treatment groups.

Groups differ at P < 0.01 with each other

42

5. DISCUSSION

5.1 Colostrum and Milk Selenium Status

The mean colostrum selenium level in the SeY group has been found below the level

(151μg/l) reported by Weiss and Hogan (2006) who supplemented the experimental

cows rations at the level of 0.3 mg selenium/kg with selenium yeast and sodium

selenite for a period of 60 days before the expected calving. This difference might be

attributed to the daily intake and basal diet selenium concentrations as the relative

increase in the colostrum selenium level of the selenium yeast group as compared to

SeI group has been found exactly the same (1.73 times) in both the studies. It is

important to note that no difference (P = 0.754) has been observed between the SeI

and control groups regarding colostrum selenium levels. These findings are also in

accordance with that of Awadeh et al. (1998a) who described no difference in

colostrums selenium content among the cows consuming approximately 0.98, 3.3

and 7.3 mg selenium/day as SeI supplement. However, their reported values in

colostrum (60-80 μg/l) are much higher than those observed in this study are. This is

probably because of the long duration of supplementation of one year before actually

taking the colostrums samples for selenium analysis. Numerous researchers agreed

that colostrum selenium content is much greater than normal milk (Abdelrahman and

Kincaid 1995; Awadeh et al. 1998a; Ortman et al. 1999; Ortman and Pehrson 1999).

It was found that selenium content in colostrum was 3.04, 2.4 and 2.54 times greater

(P < 0.05) than the average milk selenium concentrations for the control, SeY and

SeI groups respectively. These findings are in contrast with that of Weiss and Hogan

(2005) who reported 3.8 times increase (P < 0.01) in colostrum selenium

concentrations both in selenium yeast and inorganic selenium groups in their study.

In milk, a decrease of 60, 42 and 35 percent has been observed in selenium content

after one week of calving for the control, SeI and SeY groups respectively. It can be

assumed from the results of the present research that milk selenium content does not

take a steady state after about one week of milking. A sharp decreasing trend until

first week after calving is evident. However, prediction of the start of the plateau

effect in milk selenium level from this study is difficult as the next sampling was done

after ninth weeks of lactation. Up until ninth weeks after calving, the control and SeY

groups seemed to attain a plateau level whereas SeI group was not harmonious with

43

other groups in this regard. The average milk selenium content for the control, SeI

and SeY groups for all time points has been noticed as 11.6, 15.4 and 28.3 μg/l,

respectively. Milk from SeY group cows differs significantly (P < 0.05) from that of

control and SeI group cows at all time points considered in the study. On the relative

percentile scale, selenium content of the milk obtained from cows supplemented with

selenium yeast is 83 % higher than the milk from sodium selenite supplemented

cows. This finding is overall in conformance with the results of studies reviewed by

Weiss (2005), who cited a relative increase of 90%. Although milk selenium content

of the SeI group has not been found significantly different (P > 0.05) from that of

control cows, a relative increase of 32% has been noted in our study. These findings

can be explained keeping in view the previous reports that supplementation with SeI

increased milk selenium content when cows were fed rations low in naturally

occurring selenium but there was less impact when cows were fed rations greater in

naturally occurring selenium (Conrad and Moxon 1979). In addition, it was noted that

an increase in selenium intake would not produce important increases in milk

selenium content when cows were fed selenium adequate rations (Aspila 1991). A

recent systematic review (Ceballos et al. 2009) of 42 studies regarding the effect of

oral selenium supplementation on milk selenium concentrations in cattle has reported

that in Americas, selenium supplementation of 6 mg/head per day in the form of

selenium- yeast has resulted in cow’s milk selenium content of 0.37 μmol/l (30 μg/l).

Our study confirms this notion.

Higher levels of selenium in the milk of cows supplemented with selenium yeast can

be explained with the proposition that selenomethionine in selenium yeast source

replaces non-specifically methionine in the milk proteins following the genetic

sequence for the incorporation of methionine in general proteins (Ortman et al. 1999;

Weiss 2005). However, it has also been reported that only one third of total selenium

is in the form of SeMet in the milk of cows fed selenium yeast as the supplement

(Juniper et al. 2006) and therefore the possibility of presence of other selenoproteins

with antioxidant properties cannot be ruled out. It can be concluded that more work is

needed to delineate the incorporation of SeMet in different milk proteins and its effect

on their functional properties. The knowledge regarding the dairy products quality

made from the high selenium milk obtained after selenium yeast supplementation is

still limited.

44

5.2 Total Antioxidant Capacity in Milk

Milk is a rapidly perishable food commodity and development of off-flavours due to

the oxidation of various milk constituents is a major problem for the dairy industry.

Therefore, it is important to study the complex interplay of the prooxidants and

antioxidants in milk (Buettner 1993). Both fat soluble antioxidants and selenium

compounds have been implied in the protection against development of milk off-

flavour (Charmley et al. 1993; Jensen and Nielsen 1996). There is a scientific

controversy whether glutathione peroxidase activity, which is the most important

selenium-related antioxidant, is exhibited or not in milk (Chen et al. 2000; Stagsted

2006). In addition, there is lack of consensus among researchers regarding a

standardized method for the milk total antioxidant capacity estimates. Trolox

equivalent antioxidant capacity (TEAC) and oxygen radical absorbance capacity

(ORAC) are two commonly used assays for the assessment of the antioxidant

capacity of food components (Chen et al. 2003; Huang et al. 2005). We used the

former method owing to its relative simplicity and for tailoring the large number of

samples in the present study.

There is scarcity of the literature available on the topic of total antioxidant capacity as

estimated by TEAC in bovine milk. Bovine milk TEAC values of 1246 and 4560 μMol/l

measured after 10 minutes incubation time at different pH (Chen et al. 2003) 2649

μMol/l and ~5000 μMol/l measured after 3 and 20 minutes incubation time

respectively (Zulueta et al. 2009) and ~ 4600 μMol/l measured after 60 minutes

period of time (Clausen et al. 2009) have been reported. The same is the case with

milk ORAC values, which have been reported between ~5000-30000 μMol/l. There

might be many reasons for this non-conformity. It is evident that time factor is the

most important consideration in these assays. Besides the variations in the

measuring conditions, no information is available regarding feeding regime of cows

from which milk was obtained. Milk samples analysed in this study are assumed to

be low in their vitamin E content because the animals were fed below the normal

dietary requirement for vitamin E in dairy cattle (National Research Council. 2001).

One other reason for the comparatively low TEAC values might be the use of 620 nm

wavelength in the present work. The absorption maxima for the ABTS radical have

been reported as 414, 645, 734 and 815 nm (Re et al. 1999). In the present study,

45

we focussed on the issue of selenium source regarding their TEAC values in milk. It

can be indicated that the differences obtained in milk TEAC values of various

treatment groups might be attributed to their selenium content, which is significantly

higher after supplementing the cows diet with selenium yeast. Although, the same

trend has been observed in serum, more work is emphasized in this regard. This is

implicated for maintaining the milk and dairy product quality. In addition, there is need

to standardize the methods for total antioxidants capacity in milk.

5.3 Milk Selenium and TEAC Relationship

Highly significant Pearson correlation (R2 = .79; P < 0.001) has been observed

between milk TEAC values and selenium levels when data from all the groups was

subjected to statistical analysis. Regression coefficients were calculated for the

overall data and separately for the control and treatment groups (Figure 12 and Table

8). Although the overall model was found to be highly significant (P < 0.001), among

the groups only SeI model was found statistically significant in explaining the positive

correlations between TEAC and selenium level. These results further strengthen the

idea that selenium may have an effect on the milk TEAC levels. Positivity of slopes in

all models indicates that TEAC values are slightly increasing with the increase in

selenium content. These observations are contrary to that found for the serum TEAC

values, which decreased with the increase in serum selenium levels with the passage

of time. A relatively blunt slope in SeY group might be attributed to the fact that

selenium content approximately plateaus around 30 μg/l in milk. Moreover, the mean

selenium content in SeY group at 15th week of lactation was slightly less as

compared to that of 12th week. Although this novel data indicates some sort of

association between milk selenium and TEAC, further work will help delineate the

mechanisms involved.

46

Milk Se (μg/)0 10 20 30 40 50

Milk

TEA

C (μ

Mol

/l)

520

540

560

580

600

620

y = 1.55x + 533R2 = .62

Figure 12 Overall milk selenium and TEAC regression model.

Each data point corresponds to individual cows data collected at 1, 9, 12 and 15 weeks after calving.

Table 8 Regression equations describing the relationship between milk TEAC and selenium levels in various treatment groups

5.4 Milk Production

Milk production and nutrients yield data reveal that cows in experiment were normal

producers and during the experimental period selenium supplementation had no

profound effect on these parameters. No effect of selenium supplementation on the

milk yields has also been previously reported (Weiss and Hogan 2005; Bourne et al.

2008). However, in a survey conducted in Prince Edward Island (Wichtel et al. 2004),

it was noted that selenium-adequate herds had 7.6 % greater milk yield as compared

to selenium-marginal herds.

47

5.5 Serum Selenium Content in Cows

Statistical analysis of the serum selenium data in dams has revealed that there is no

significant difference between SeI and SeY groups. However, both treatment groups

differ from the control cows. These findings seem to be in conformance with that of

Juniper et al. (2006) who reported no difference (P > 0.05) in whole blood selenium

levels of cows with total dietary selenium intake from selenium yeast and sodium

selenite of 6.34 and 5.85 mg/day. The lack of significant differences between sodium

selenite and selenium yeast in raising the blood or serum selenium levels can be

associated with the dietary selenium intake. Significant difference was observed

when cows were consuming 0.24-0.31 mg selenium/kg DM (Knowles et al. 1999;

Ortman and Pehrson 1999). The relative increase in serum selenium levels of SeY

group cows found in this study (8-34%) is in agreement with whole blood selenium

levels in other studies reviewed (Weiss 2005). It is interesting to point out that serum

selenium levels in the control cows are far below the reference values (70 μg/l)

reported as indication of the adequate selenium status in dairy herds (Stowe and

Herdt 1992) notwithstanding that dietary selenium intake from the basal diet (Table 3)

was in accordance with the German recommendations (GfE 2001). These findings

strengthen the results obtained previously in Germany (Gierus et al. 2002) reporting

plasma selenium levels of 37.7μg/l with dietary selenium intakes up to 0.165 mg/kg

DM. It can be speculated that cows might be at the risk of deficiency or at least

vulnerability to disease threat. This provides a base for reconsideration of selenium

dietary recommendation for dairy cows.

Decrease in the serum selenium levels at calving can be attributed to selenium

transfer to calves. A relative increase of 30% has been noted in the calves born to

dams of the SeY group as compared to ones in SeI group. This difference (30%) has

not been found significant (P = 0.33) and is less (37%) than that observed by Weiss

and Hogan (2005). However, one week after being fed on colostrums of their dams, a

significant increase (P < 0.05) of 26 % has been noted in calves of SeY group when

compared with those in SeI group. Again, this difference is comparatively less than

reported previously regarding whole blood of calves (Awadeh et al. 1998a; Gunter et

al. 2003). Possible reasons for this might be long supplementation duration in

previous studies and high supplementation doses in the present investigation.

48

5.6 Selenium Transfer from Cows to Calves

Relationships between serum selenium levels of dams and their calves on the day of

calving and one week after calving have been determined by regression analysis

(Table 9). Overall regression model for all the groups has been presented in the

Figure 13. It is evident that comparatively stronger relationship (R2 = .57, P < 0.01)

exists in the control group. This supports the idea that response to supplementation

is weaker beyond the undefined threshold levels (Juniper et al. 2006). SeY group is

also stronger (R2 = .41, P < 0.05) than SeI group in transferring the dams selenium to

calves.

Cows serum Se level (μg/l)

0 20 40 60 80 100 120 140

Cal

ves

seru

m S

e le

vel (

μg/l)

0

10

20

30

40

50

60

y = 0.3394x + 15.23R2 = .576

Figure 13 Regression model describing the relationship between dams and calves serum selenium levels.

Each data point corresponds to individual cows and calves data collected at calving and 1 week after calving.

49

Table 9 Regression equations describing the relationship between calves and dams serum selenium levels in various treatment groups

5.7 Serum Trolox Equivalent Antioxidant Capacity (TEAC) in Cows

The results of Trolox equivalent antioxidant capacity in serum have been found to

act in accordance with those observed in the milk regarding the treatment effect. On

the other hand, TEAC values in milk and serum differ with respect to the time point

effect. Decreasing trend in TEAC values in serum, contrary to milk, has been noted.

Regression analysis (Table 10 )shows that slight negative correlations exist between

the serum selenium and TEAC values within treatment groups, however, these

models have been found non-significant with very low R2 values in explaining the

negativity of the slopes contrary to the overall model with stronger values (R2 = .406;

P < 0.001). Within the groups, SeY model seems to be comparatively stronger

because selenium content in serum increased more sharply than other groups.

This data is novel as no previous report describes TEAC in serum of cows and

calves. Although a strong indication of the effect of selenium on the total antioxidant

capacity can be noted, further work in this regard can delineate the actual

mechanism involved. It can be speculated from the contrasting findings in milk and

serum regarding the effect of time on TEAC values, that different selenoproteins or

selenium-containing proteins might be present in milk and serum.

50

Table 10 Regression equations describing the relationship between serum selenium and TEAC in dams

Equation Group n R2 Slope SE Intercept SE P <

1 Overall 48 .406 0.120 0.021 564.29 1.45 0.001

2 Control 15 .088 -0.044 0.039 567.23 1.51 0.282 3 SeI 15 .008 -0.004 0.013 571.02 0.93 0.749 4 SeY 15 .091 -0.011 0.007 578.64 0.54 0.117

Dependent variable = TEAC (μMol/l); Independent variable = selenium (μg/l)

Serum Se (μg/l)0 20 40 60 80 100 120 140

Ser

um T

EAC

(μM

ol/l)

555

560

565

570

575

580

585

y = 0.1203x + 564R2 = .406

Figure 14 Regression model describing the relationship between serum selenium and TEAC in dams.

Each data point corresponds to individual cows’ data collected at calving, 1 and 12 weeks after calving

51

5.8 Serum TEAC in Calves

Serum TEAC values in calves have been noted to follow a similar pattern regarding

the treatment effect as noted in dams. Regression analysis (Figure 15) with selenium

and TEAC values data has also generated a model quite similar to that for dams.

Serum Se (μg/l)0 10 20 30 40 50 60

Ser

um T

EA

C (μ

Mol

/l)

562

564

566

568

570

572

574

576

578

580

y = 0.201x + 562R2 = .396

Figure 15 Regression model describing the relationship between calves’ serum selenium and TEAC values.

Each data point corresponds to individual calves data collected within 12 hrs after calving and 1 week after calving

52

6. Trace Element Status in Large Dairy Herds

This work was supported by a grant from Sächsisches Landesamt für Umwelt,

Landwirtschaft und Geologie. The author acknowledges the cooperation and support

rendered by Dr. Steinhöfel and Mrs. Fröhlich.

6.1 Introduction

Scientific research over a long period has proved that many minerals are essential

for the normal growth, physiological functioning and productivity of ruminants. Among

these are included macro and micro minerals. Provision of adequate levels of trace

metals in cattle diet is essential to promote growth and maintain animals in good

health (Blanco-Penedo et al. 2009). Trace elements such as copper, zinc,

manganese, iron and selenium are most important micro minerals for dairy cattle

which are not only essential for the well being of the animals themselves but also

have an importance for the public health owing to their transfer to them through the

milk. As milk is an important component of the daily diets of human beings of all

ages, deficiencies of these essential nutrients in dairy cows rations and consequently

in the milk are likely to be reflected in the human populations. Moreover, trace metals

that are included as mineral supplements may have toxic effects at supra-optimal

concentrations (Underwood and Suttle 2002).

A number of ways can be applied to diagnose the possible deficiencies.

Development of clinical symptoms and identification of post mortem tissue lesions

can give some clue in this regard. However, differential diagnosis of any particular

trace elements deficiency will be difficult because most of them do not show unique

clinical signs or lesions for deficiency. In other instances, indirect proof of the

deficiency can be provided by the positive response to supplementation of the

suspected deficient mineral. This may not be incorrigible as time responsive effects

of clinical signs might occur. It has been noted that trace elements are embedded in

trace enzymes (Köhrle 2000). Although a difficult approach especially when large

herds are concerned, the best way to establish the deficiency of a trace element is by

testing for the unique functional deficit or the deficiency of the specific mineral

containing protein or enzyme.

53

Estimation of the trace elements in various animal tissues of the representative

samples can give an indication of the herds’ mineral status. Usually the liver biopsy

samples, whole blood or serum, milk, urine and hair samples can be used for the

purpose. Liver biopsy is technically demanding in large populations of animals on the

fields (Guyot et al. 2009). Plasma has been described as the most commonly used to

assess copper and zinc status (Kincaid 2001). However, care must be taken about

the feeding regimen, supplementation routine, disease condition and proper number

of samples and standard procedures (Maas 2007) while making tissue analysis a

criterion for the herd mineral status. Different antagonistic and positive correlations

must also be kept in mind.

A survey of 11 selected farms concerning copper, zinc, manganese, iron and

selenium was conducted to assess the trace element nutritional status of dairy cows

and subsequent levels in liver and plasma samples. The objective was to study the

intake, bioavailability and interactions among essential trace elements in large dairy

herds under prevalent feeding practices. The impact on herd health was also a

consideration.

6.2 Farms, Animals and Sampling

A survey of selected trace elements (copper, zinc, manganese, iron and selenium)

was conducted for 11 large commercial dairy herds maintained in the state of

Saxonia, Germany. The project was carried out with the support of Sächsisches

Landesamt für Umwelt, Landwirtschaft und Geologie (Köllitsch). Liver biopsies and

plasma samples were taken from 10 selected animals of each dairy farm. Twenty

(20) samples of TMR were taken and analysed by the Landesamt for the whole

nutrient composition and the data obtained were used for further statistical analysis.

Liver biopsies and plasma samples were analysed for five selected trace elements

composition at the Institute of Animal Nutrition laboratory, Freie Universität Berlin,

using atomic absorption spectrometry (Vario 6 equipped with H52 hydride system

and auto sampler, Analytik Jena AG, Germany). Analytical methods were

standardised using reference standards (Atomic Spectroscopy Quality Control

Standard 21, Perkin Elmer) and the results were within the permissible range (Figure

16). Plasma biochemistry was analysed by the Landeslabor Berlin-Brandenburg,

Berlin.

54

Trace elements

Fe Cu Zn Mn

mg/

l

0

20

40

60

80

100

120

Figure 16 Trace elements results measured compared to a standard

value of 100 mg/l (n=3)

6.3 Statistical Analysis

Data were statistically analysed using SPSS 15 for descriptive statistics and various

correlations. Multiple linear regression models were found out regarding the

interaction of trace elements in feeds and liver tissue. Stepwise regression method

was followed for multiple regression analyses in which feed trace elements, feed

minerals and the remaining nutrient composition was added at consecutive stages.

Level of significance was set as P < 0.05. Kolmogorov-Smirnov (KS) test was run to

determine the normality of data.

6.4 Results and Discussion

Descriptive statistics for the data obtained on feed composition from 11 commercial

dairy farms included in the survey has been summarized in the following Table 11.

Proximate constituent data reveal measured nutrients mean values fall within the

normal range according to the recommended dietary allowances. However, great

farm-to-farm variation can be noted. This is attributable to different farm practices.

The data seem to meet the assumption of normality when subjected to KS test.

Macro minerals mean values have also been found to be little deviating from the

recommended dietary allowances for dairy cattle. It can be noted that large variation

in the sodium and chloride content has rendered the data non-normal. This might

55

have an effect on the dietary cation anion balance and subsequent productive

performance in the concerned herds. Dietary cation anion balance calculated from

the mean values for TMR is +17 milliequivalents that is quite less than the normal

range (+ 20 to + 40 mEq/100 g dietary DM) described for rations for the lactating

dairy cows (Beede 2005).

It is interesting to point out that all trace elements measured in TMR were found to be

more than the recommended dietary allowances. Iron content of the ration has been

noted to be exceptionally high. Overall variation in the trace element composition of

the diet was also high (coefficient of variation, 22% - 43%). This indicates the high

dosage use of mineral supplementation by the farmers or problems with mixing and

homogeneity during the ration preparation could occur.

Table 11 Descriptive summary of feed composition (DM basis) data collected from 11 different farms in Saxonia (Germany)

Number Nutrient Mean* Range SEM KS statistics **

Proximate Constituents 1 Dry matter g/kg 414.1 320.7 - 487.5 2.9 .720 2 Crude ash g/kg 69.3 56.3 - 85.9 0.46 .294 3 Crude protein g/kg 170.4 137.4 - 213.9 1.04 .765 4 Crude fibre g/kg 165.6 141.2 - 204.0 0.91 .763 5 Crude fat g/kg 43.4 30.4 - 55.5 0.42 .941 6 Starch g/kg 228.3 159.3 - 276.2 1.82 .974 7 Sucrose g/kg 44.0 11.9 - 93.4 1.36 .0558 Soluble organic

matter g/kg 754.6 664.7 - 792.4 1.71 .43

Mineral Constituents 9 Calcium g/kg 7.2 3.9 -10.2 0.09 .238 10 Phosphorus g/kg 4.2 3.1 - 5.1 0.03 .833 11 Sodium g/kg 1.8 0.3 - 9.3 0.09 <.00112 Magnesium g/kg 2.4 1.7 -3.1 0.02 .008 13 Potassium g/kg 14.3 9.8 - 20.1 0.15 .743 14 Sulphur g/kg 2.1 1.7 - 2.7 0.01 .356 15 Chloride g/kg 4.6 1.9 -18.9 0.18 .00116 Copper mg/kg 23.9 9.4 - 44.5 0.56 .331 17 Zinc mg/kg 97.1 42.0 -163.0 2.31 .227 18 Manganese mg/kg 71.9 32.4 - 119.1 1.63 .285 19 Iron mg/kg 374.6 208.1 - 655.4 6.17 .419 20 Selenium mg/kg 0.5 0.06 - 1.2 0.02 .696

*Mean values obtained after the analysis of 200 samples **Kolmogorov-Smirnov (KS test) statistical values > 0.05 meet the assumption of the normality of the data

56

The whole feed data from 11 farms were subjected to Principal Component Analysis

(PCA). The purpose of PCA is to express the main information contained in the initial

variables in a lower number of variables, the so-called principal components (latent

variables), which describe the main variations in the data. Practically PCA transforms

a number of possibly correlated variables in a smaller number of uncorrelated

variables or principal components. This statistics helps to perform the multiple

regression analysis in situations where a large number of independent variables

might have a cumulative effect on the dependent variable. By applying PCA on the

feed composition data from the Saxonian dairy herds, it is observed that trace

elements manganese, zinc, copper and selenium fall within the same principal

component one. This means this component might have an effect as a group on the

trace element concentrations in the liver or plasma or any other parameter of interest.

Moreover, this also shows a trend of supplementation of these minerals. These latent

variables generated could further be used in simple or multiple regression analysis.

This is a novel result and application of this statistical tool to large sets of feed data

should be further studied to find out interactions and relationships among various

nutrients. Following table 12 and the related scree plot diagram shows a distinct

group of trace elements with very high loadings in the component 1.

Table 12 Component matrix resulted from the principal component analysis of feed data of 11 dairy herds

Feed Factors Extracted 1 2 3 4

Zinc 0.919 Sodium 0.815

Manganese 0.815 Selenium 0.782 Copper 0.777 Sulphur 0.920

Crude Protein 0.910 Phosphorus 0.441 0.757 Magnesium 0.527 0.613 Potassium 0.901

Starch -0.804 Crude Ash 0.787 Dry Matter 0.889 Sucrose 0.726

Extractions method: Principal component analysis Rotations method: Varimax with Kaiser-Normalisation Kaiser Meyer Olkin = 0.70 Bartlett test of sphericity P < 0.001, (df = 91)

57

Figure 17 Screeplot diagram of the feed components

Milk production, plasma biochemistry and the liver trace elements data have been

summarised in the following Table 13. It is evident that milk production data for two

consecutive months did not much differ. Plasma biochemistry parameters reveal the

cows to be healthy.

Liver tissue concentrations of trace elements are subjected to changes, depending

on the age, production stage and disease condition of the animal and might exhibit

large variations. Zinc liver content in this study has been found lower than recently

reported (Nriagu et al. 2009) as 29.5 mg/kg (fresh weight) in grazing dairy cows.

However, copper concentration in our study (134.5 mg/kg fresh weight) has been

found quite high as compared to 20.4 mg/kg in the study of Nriagu et al. (2009). A

previous report described a range of 1.4 – 134.5 mg/kg fresh weight in grazing cattle

in Queensland, Australia (Kramer et al. 1983). Whereas, mean selenium

concentration are relatively close to each other (0.43 mg/kg ~ 0.72 mg/kg). These

findings indicate an expected lower content of trace element in the grazing cows,

which can be due to the lower soil content. Contrary result regarding zinc might be

associated with some particular antagonistic relationships.

58

Table 13 Descriptive summaries of various parameters measured in samples collected from 11 different farms in Saxonia (Germany)

Nutrient Mean* Range SEM KS statistics

** Milk November 2008

Milk yield (kg) 38.02 20.60 – 59.30 0.69 .905 Fat (%) 3.76 2.0 – 5.75 0.07 .868 Protein (%) 3.37 2.65 – 4.14 0.03 .908 Milk urea (mg/l) 245.54 130 - 380 4.60 .230 Somatic cell count * 1000 229 9 - 3624 54.55 .000Lactose (%) 4.80 4.22 – 5.17 0.02 .183 Days in milk 122 56 - 297 3.34 .383 Protein corrected milk (kg) 36.79 20.90 – 64.20 0.64 .765

Milk December 2008 Milk yield (kg) 36.11 20.20 – 54.90 0.65 .924 Fat (%) 3.94 2.63 – 5.89 0.06 .998 Protein (%) 3.45 2.81 – 4.18 0.03 .946 Milk urea (mg/l) 257.72 140 - 430 4.86 .820 Somatic cell count * 1000 159 4.92 – 24.17 35.02 .000 Lactose (%) 4.77 4.22 – 5.28 0.02 .169 Days in milk 154 91 - 321 3.31 .326 Protein corrected milk (kg) 35.84 19.20 – 50.30 0.59 .940

Plasma Biochemistry ALAT (μkat/l) 0.84 0.55 – 1.18 0.01 .423 ASAT (μkat/l) 1.89 0.89 – 3.26 0.05 .059 Bilirubin (μmol/l) 4.16 1.40 – 8.10 0.09 .065 Bilirubin indirect (μmol/l) 4.02 0 – 7.80 0.01 .015 Bilirubin direct (μmol/l) 0.13 0 – 0.50 0.10 .000 Ferritin (ng/l) 30.97 3.80 – 161.40 2.00 .047

Plasma Trace Elements Zinc (mg/l) 1.51 (n=94) 0.39 - 2.77 0.06 .195 Copper (mg/l) 1.78 (n=106) 0.18 - 8.09 0.15 .002Manganese (mg/l) 0.80 (n=54) 0.02 - 4.20 0.13 .642 Selenium (mg/l) 0.101 (n=106) 0.05 – 0.17 1.85 .275 Iron (mg/l) 1.71 (n=66) 0.09 - 6.07 0.13 .355

Liver Trace Elements (Fresh matter basis)Zinc (mg/kg) 18.26 (n=106) 1.5 – 46.7 0.90 .185 Copper (mg/kg) 134.58 (n=105) 1.4 – 372 6.81 .85 Manganese (mg/kg) 7.22 (n=106) 0.29 – 52.5 0.71 .001 Iron (mg/kg) 89.17 (n=108) 3 – 278 6.35 .033 Selenium (mg/kg) 0.721 (n=104) 0.122 – 1.55 0.04 .272

*Mean values obtained after the analysis of 110 samples, for trace elements n is specified in brackets **Kolmogorov-Smirnov statistical values > 0.05 meet the assumption of the normality of the data

59

Multiple correlations and regression models among trace elements in feeds and liver

tissue have been sorted out. The results are presented in the Table 14 and Table 16.

Pearson correlation matrix among the liver trace elements (Table 14) clearly

indicates strong positive and negative correlations among various trace minerals.

zinc and copper have been shown to exhibit significant positive correlations with

manganese and selenium liver contents and with each other. Strong positive

correlation (R = .35, P < .001) between zinc and copper are comparatively higher

than previously reported (R = .19, P = .004) by Nriagu et al. (2009).

It can be noted that high iron concentrations in the liver are having strong negative

correlations with all other trace elements in the liver. iron, sulphur, molybdenum and

stress have been described as the antagonists to copper, zinc and manganese

bioavailability in dairy cows (Nockels et al. 1993).

Table 14 Pearson correlation matrix for various trace elements in liver tissues

Zinc Copper Manganese Selenium Liver Zinc R = .353 R = .358 R = .229

P <0.001 P <0.001 P = 0.018 N = 104 N = 104 N = 106

Copper R = .353 R = .227 R = .613

P < 0.001 P =0 .021 P <0.001N = 104 N = 102 N = 105

Manganese R = .358 R = .227 R = .008

P = 0.001 P =0.021 P =0.937 N = 104 N = 102 N = 104

Selenium R = .229 R = .613 R = .008

P = 0.018 P <0.001 P = 0.937 N = 106 N = 105 N = 104

Iron R = -.433 R = -.503 R = -.257 R = -.266 P < 0.001 P <0.001 P = 0.008 P = 0.006 N = 105 N = 104 N = 105 N = 106

When all data on the feed composition and trace mineral in liver tissues were

subjected to multiple linear regression analysis in a stepwise method, models were

generated (Table 16). It is evident that trace elements included in the analysis have

been found to be interacting with one and other in positive and negative relationships.

60

Selenium, copper and manganese in the feed have been found to increase their

respective liver concentration whereas zinc and iron have negative relationship.

Overall, it can be concluded that trace elements in feeds show antagonism towards

one another of various magnitude. Possible reasons could be the chemical affinity of

transition metals towards various biomolecules in the physiological system of

ruminants. More work is ascertained in this regard.

Feed (μg/kg DM)

200 400 600 800

Live

r (μg

/kg

fresh

mat

ter)

400

600

800

1000

1200

1400

1600

Se

P = 0.095

Figure 18 Feed and liver selenium relationship in Saxonian dairy farms

61

Feed (mg/kg DM)

40 60 80 100 120 140 160

Live

r (m

g/kg

fres

h m

atte

r)

0

5

10

15

20

25

30

35

Zn

P = 0.029

Figure 19 Feed and liver zinc relationship in Saxonian dairy farms

Feed (mg/kg DM)

10 15 20 25 30 35 40

Live

r (m

g/kg

fres

h m

atte

r)

60

80

100

120

140

160

180

200

220

Cu

P = 0.28

Figure 20 Feed and liver copper relationship in Saxonian dairy farms

62

Feed (mg/kg DM)

200 300 400 500

Live

r (m

g/kg

fres

h m

atte

r)

20

40

60

80

100

120

140

Fe

P = 0.685

Figure 21 Feed and liver iron relationship in Saxonian dairy farms

Feed (mg/kg DM)

40 60 80 100 120

Live

r (m

g/kg

fres

h m

atte

r)

0

2

4

6

8

10

12

14

16

18

Mn

P = 0.406

Figure 22 Feed and liver manganese relationship in Saxonian dairy farms

63

Table 15 Regression equations describing relationship between feed and liver tissues concentrations of various trace elements

Trace Element R R2 F Ratio Constant Coefficient t p

Selenium .524 .274 3.40 360.11 888.66 1.84 0.095Zinc .653 .427 6.70 33.36 -.15 -2.58 0.029Copper .358 .128 1.32 83.59 2.13 1.15 0.280Manganese .279 .078 0.76 11.69 -.06 -.87 0.406Iron .138 .019 .175 109.81 -.06 -.41 0.685

Table 16 Multiple linear regression models describing the relationship among liver trace elements (dependent) and feed composition (independent)

Dependent variable

Independent variables

R2 F Ratio p Standardised Coefficient t p

Selenium .960 12.03 0.033 -9.658 2.99 0.040Se 1.702 5.49 0.012Cu 0.118 0.47 0.688 Zn -0.773 -2.10 0.127 Mn -0.818 -2.46 0.091 Fe -0.280 -1.73 0.182 ELOS 0.799 3.35 0.044

Zinc .883 6.06 0.053 53.310 3.94 0.017Zn -0.913 -1.78 0.149 Cu -0.309 -1.32 0.255 Mn -0.214 -0.70 0.523 Fe 0.076 0.32 0.766 Se 0.494 1.10 0.333

Copper .960 11.86 0.034 -100.340 -1.67 0.193 Cu 0.018 0.11 0.919 Zn -.673 -1.93 0.149 Mn -1.026 -4.15 0.025Fe 0.064 .39 0.723 Se 0.731 2.23 0.111 Crude Ash 0.959 6.22 0.008

Manganese .984 30.49 0.009 -3.340 -0.46 0.676 Mn 0.155 0.87 0.450 Fe 0.189 1.42 0.251 Se -0.173 -0.89 0.435 Zn -0.312 -1.25 0.297 Cu -0.471 -4.45 0.021K 0.818 6.86 0.006

Iron .998 176.91 0.006 258 14.23 0.005Fe -0.535 -6.34 0.024Se 0.476 5.13 0.036Zn -0.576 -4.59 0.044Cu 0.543 13.11 0.006Mn 0.789 11.21 0.008K -0.378 -4.16 0.053 Crude Ash -0.373 -5.03 0.037

64

Data on various parameters of milk production and trace element concentrations in

liver were subjected to multiple linear regression analysis to find out the relationships

among these variables. The results have been presented in Table 17. It can be noted

that liver copper concentrations are having strong positive impact on the daily milk

yield whereas zinc concentration are negatively correlated to milk yield. Somatic cell

count has also been found to be negatively related with milk yield. This is according to

Rainard and Riollet, (2006) who described that SCC decreased with the progress in

lactation. Age of the cows has an effect on the SCC. With the increase in age, SCC in

milk is also increased. This is logically attributable to the wear and tear in mammary

gland tissues occurred with the increase in age. It is also evident from the strong

positive correlation (R = .365; P < 0.001) noted between SCC and the age of the

animal.

Table 17 Multiple linear regression models describing the relationship among liver trace elements and various parameters of herd performance

*Beta in (variables, which can be included in the original model with these values)

65

7. CONCLUSION The research carried out thus far regarding the possible role of selenium in dairy cow

nutrition has been primarily focused on the broad areas of effects of organic and

inorganic selenium sources on selenium status, bioavailability and transfer to

offspring and GSHPx activities and subsequent impact on the immune function. Still

there are gaps in our present set of knowledge, which should be filled. The lower limit

of 0.2 ppm of selenium in diets is generally regarded as a level below which the

immune system might be vulnerable. Although, in our study no adverse health effects

in the control group could be noted, the significantly lower selenium levels in serum,

milk and selenium transfer to offspring compared to the other groups may pretend

some sort of risk. Previous findings about significant increases in the milk and serum

selenium levels as a result of the selenized yeast supplementation in the diets have

been strengthened with the results of the present study. Although distribution of

selenium in different milk and protein fractions has been worked out, our knowledge

regarding the presence and characterization of selenoproteins in milk and their

effects on dairy consumers and possible influence on technical properties of dairy

products is still poor. Mechanisms behind the positive impact of selenium yeast on

the total antioxidant capacity as observed in this study and the expected

improvement in the milk quality are not fully elucidated. It can be argued that

selenium might be interacting with the fatty acids present in the milk.

Survey findings have revealed a general trend of over supplementation for trace

minerals in dairy rations. Minerals antagonistic relationships must be considered

while formulating rations.

Important areas for further research and recommendations on the topic are following:

Dietary recommendations for selenium in dairy cows rations should take into

consideration different bioavailability of selenium sources, however, more data

are needed considering the biological function

Interactions with other micronutrients (copper, zinc, manganese and iron) and

mammary gland trace element homeostasis should be studied further

Milk selenoproteins and selenium-containing proteins should be more extensively

characterised

Standardised methods for total antioxidant capacity are needed to be established

66

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83

APPENDIX 1 Data on Feeding Trial with Dairy Cows Conducted at BfR

Serum Se content (µg/l) in cows

Cows Weeks relative to calving SeI Feeding - 6 W - 3 W Birth 1 W 12 W 321 6.12.08 75.23 84.39 68.52 88.27 83.65 338 18.9.08 46.39 56.91 60.66 69.55 97.45 340 26.10.08 31.00 47.83 48.83 67.58 49.56 357 30.5.08 51.68 62.31 39.52 60.35 74.04 361 16.7.08 42.12 60.87 42.98 74.91 58.48 SeY 322 26.1.2009 66.39 75.24 68.07 86.19 126.10 349 27.5.08 31.31 47.96 35.78 67.00 55.35 353 18.9.08 56.12 59.08 67.85 85.35 122.30 355 30.12.08 62.59 74.76 62.85 76.67 85.18 359 16.7.08 43.97 70.76 47.98 76.22 96.72 412 24.11.08 66.41 78.95 89.06 118.30 99.06

Control 311 27.5.08 43.92 38.40 12.32 36.69 20.04 351 2.6.08 50.95 30.14 9.10 34.92 25.98 352 27.5.08 28.40 26.50 23.44 33.00 29.03 360 2.8.08 51.31 51.32 56.35 14.94 58.39 431 26.1.09 64.90 46.16 51.65 56.26 54.55

Serum TEAC values (µMol/l) in cows

Cows Weeks relative to calving SeI Feeding Birth 1 W 12 W 321 6.12.08 571.48 571.68 570.36 338 18.9.08 572.66 570.96 569.91 340 26.10.08 570.46 570.18 569.46 357 30.5.08 571.13 570.63 569.81 361 16.7.08 571.26 570.86 570.06 SeY 322 26.1.2009 578.03 578.06 582.38 349 27.5.08 577.53 577.51 576.58 353 18.9.08 577.11 577.16 576.66 355 30.12.08 577.43 577.76 576.73 359 16.7.08 577.73 578.41 576.63 412 24.11.08 577.71 577.61 576.08

Control 311 27.5.08 566.48 566.73 565.61 351 2.6.08 566.48 567.11 565.53 352 27.5.08 567.46 566.86 565.16 360 2.8.08 566.41 566.06 565.78 431 26.1.09 566.96 566.33 556.63

84

Calves born to the experimental cows Calves Sex Serum Se (µg/l) Serum TEAC (µMol/l)

SeI Birth 1 W Birth 1 W 321 F 44.06 43.10 571.76 570.13 338 M 32.92 37.17 571.73 570.36 340 F 29.08 31.32 571.96 570.46 357 F 10.20 39.37 561.21 561.21 361 dead SeY 322 F 44.42 48.85 578.56 577.28 349 M 36.85 41.20 577.33 576.96 353 M 31.30 43.32 578.38 577.16 355 M 40.36 53.15 578.53 577.18 359 dead 412 M 37.33 51.96 567.81 567.81

Control 311 M 20.83 41.54 568.13 566.13 351 F 8.09 26.69 567.66 566.48 352 F 23.72 30.61 567.93 566.53 360 F 32.75 13.14 567.76 566.81 431 M 32.19 29.82 567.51 566.31

Milk TEAC values (µMol/l) in cows

Cows Weeks relative to calving SeI 1 W 9 W 12 W 15 W 321 557.99 558.69 557.44 557.89 338 555.89 557.64 557.64 558.64 340 556.69 557.49 557.69 557.44 357 557.34 557.54 557.49 557.54 361 556.49 557.94 557.64 557.94 SeY322 585.82 586.97 587.25 586.72 349 586.07 586.42 586.62 586.00 353 586.25 587.05 587.37 587.42 355 584.17 586.82 587.05 588.15 359 585.62 585.87 586.42 587.57 412 586.32 587.57 587.50 587.85

Control311 540.20 541.55 541.47 542.00 351 540.80 540.90 542.22 543.62 352 539.65 540.47 540.75 540.97 360 540.57 541.27 541.57 541.50 431 540.02 541.22 541.62 541.20

85

APPENDIX 2 Saxonian Dairy Herds Data – Feed Composition Fa

rm 1

Nutrients N Minimum Maximum Mean SEM Dry matter g/kg 19 388.10 465.35 427.87 5.97Crude ash

g/kg

DM

19 56.90 63.52 60.08 0.55Crude protein 19 163.22 191.47 177.58 1.91ELOS 19 595.12 775.76 733.67 12.24Crude fibre 19 140.99 186.85 163.80 3.12Crude fat 19 34.18 62.32 45.33 1.81Starch 19 218.12 249.24 235.30 2.11Sucrose 19 27.19 48.03 36.37 1.57Calcium 19 4.52 8.61 6.34 0.27Phosphorus 19 4.03 5.16 4.55 0.06Sodium 19 0.64 1.61 1.10 0.07Magnesium 19 2.10 3.17 2.62 0.08Potassium 19 12.01 14.21 13.05 0.17Sulphur 19 2.00 2.69 2.33 0.04Chloride 19 1.79 3.49 2.49 0.11Copper

mg/

kg D

M 19 9.66 39.70 20.99 2.01

Zinc 19 40.75 173.46 91.58 9.46Manganese 19 32.03 71.37 49.09 2.85Iron 19 249.03 374.65 290.27 6.75Selenium 19 0.15 0.76 0.46 0.05

Farm

2

Dry matter g/kg 21 367.71 483.75 412.97 5.27Crude ash

g/kg

DM

21 58.56 77.37 68.45 0.91Crude protein 21 128.29 214.73 184.46 4.08ELOS 21 706.22 764.05 740.00 3.29Crude fibre 21 161.52 193.65 176.72 2.06Crude fat 21 31.13 43.74 38.95 0.64Starch 21 173.73 241.98 208.02 4.48Sucrose 21 43.85 61.64 51.26 1.23Calcium 21 5.37 10.55 8.01 0.30Phosphorus 21 3.25 4.95 4.29 0.08Sodium 21 0.29 1.17 0.75 0.06Magnesium 21 1.99 2.62 2.31 0.03Potassium 21 13.16 18.36 15.35 0.29Sulphur 21 1.99 2.73 2.37 0.04Chloride 21 1.79 4.26 3.07 0.14Copper

mg/

kg D

M 21 8.53 47.21 28.32 2.51

Zinc 21 38.34 135.09 84.52 6.21Manganese 21 33.81 92.61 65.60 3.71Iron 21 285.90 472.28 387.97 9.46Selenium 21 0.07 0.81 0.44 0.05

86

Fa

rm 3

Nutrients N Minimum Maximum Mean SEM

Dry matter g/kg 20 433.27 485.59 468.90 3.66Crude ash

g/kg

DM

20 72.68 83.37 77.32 0.75Crude protein 20 163.32 194.58 183.40 1.69ELOS 20 731.27 779.19 756.52 2.68Crude fibre 20 142.98 169.57 154.52 1.54Crude fat 20 36.61 42.58 40.47 0.36Starch 20 200.63 227.16 212.43 1.72Sucrose 20 75.49 95.74 86.94 1.30Calcium 20 6.62 8.62 7.86 0.11Phosphorus 20 4.16 4.97 4.62 0.05Sodium 20 1.00 1.43 1.25 0.03Magnesium 20 2.46 3.06 2.84 0.03Potassium 20 16.16 18.06 17.02 0.09Sulphur 20 2.13 2.58 2.40 0.03Chloride 20 3.60 5.02 4.10 0.08Copper

mg/

kg D

M 20 18.15 23.88 21.14 0.37

Zinc 20 70.24 99.64 84.11 1.93Manganese 20 46.40 63.81 53.34 0.92Iron 20 269.32 559.43 348.17 14.03Selenium 20 0.21 0.50 0.35 0.02

Farm

4

Dry matter g/kg 21 414.55 460.49 432.72 2.92Crude ash

g/kg

DM

21 62.13 74.76 66.13 0.73Crude protein 21 144.36 177.73 159.17 1.92ELOS 21 760.37 796.01 781.68 2.41Crude fibre 21 133.19 169.67 150.09 1.99Crude fat 21 30.39 43.51 34.82 0.78Starch 21 206.68 283.57 248.65 4.06Sucrose 21 36.25 78.26 55.51 2.86Calcium 21 5.20 9.20 6.82 0.24Phosphorus 21 3.18 4.45 3.70 0.06Sodium 21 1.00 1.93 1.30 0.05Magnesium 21 1.70 2.55 2.03 0.04Potassium 21 11.68 17.76 14.63 0.33Sulphur 21 1.99 2.55 2.19 0.03Chloride 21 2.43 5.20 3.21 0.13Copper

mg/

kg D

M 21 11.19 23.97 14.40 0.81

Zinc 21 40.28 87.59 52.97 2.52Manganese 21 39.33 76.88 50.87 1.80Iron 21 324.49 511.22 400.79 11.57Selenium 21 0.08 0.53 0.20 0.02

87

Farm

5

Nutrients N Minimum Maximum Mean SEM Dry matter g/kg 20 314.91 452.45 380.41 7.78Crude ash

g/kg

DM

20 61.49 79.55 75.02 1.04Crude protein 20 133.19 222.28 160.00 4.18ELOS 20 706.78 811.92 751.72 5.26Crude fibre 20 124.79 212.70 177.94 4.45Crude fat 20 36.49 60.22 42.20 1.07Starch 20 131.30 263.96 203.42 6.47Sucrose 20 24.18 50.34 34.76 1.75Calcium 20 5.41 7.43 6.58 0.11Phosphorus 20 3.50 5.42 4.11 0.09Sodium 20 0.88 1.88 1.47 0.05Magnesium 20 1.85 2.55 2.21 0.04Potassium 20 11.68 24.06 16.33 0.52Sulphur 20 1.59 2.76 2.01 0.05Chloride 20 2.72 4.24 3.73 0.10Copper

mg/

kg D

M 20 16.54 29.55 23.62 0.78

Zinc 20 60.98 109.06 83.30 2.85Manganese 20 59.17 98.24 77.46 2.17Iron 20 283.40 444.14 358.61 10.94Selenium 20 0.22 0.62 0.40 0.03

Farm

6

Dry matter g/kg 20 369.78 400.68 383.49 1.93Crude ash

g/kg

DM

20 69.13 83.32 72.68 0.73Crude protein 20 157.03 195.36 177.86 2.04ELOS 20 706.53 751.72 733.42 2.26Crude fibre 20 158.60 198.02 176.13 1.71Crude fat 20 40.11 48.93 45.71 0.49Starch 20 147.16 213.13 192.01 3.13Sucrose 0 Calcium 20 6.69 7.85 7.30 0.07Phosphorus 20 4.57 5.35 5.01 0.05Sodium 20 2.34 3.04 2.72 0.04Magnesium 20 2.76 3.29 3.01 0.03Potassium 20 12.86 18.09 14.75 0.26Sulphur 20 2.02 2.46 2.24 0.02Chloride 20 5.31 7.13 6.22 0.10Copper

mg/

kg D

M 20 34.41 42.62 38.56 0.49

Zinc 20 133.71 165.36 153.14 1.91Manganese 20 91.65 118.76 107.37 1.49Iron 20 314.43 420.41 343.90 5.89Selenium 20 0.33 0.55 0.46 0.02

88

Farm

7

Nutrients N Minimum Maximum Mean SEM Dry matter g/kg 22 338.14 383.33 364.73 2.88Crude ash

g/kg

DM

22 59.71 83.29 74.33 1.07Crude protein 22 150.96 178.63 166.70 1.54ELOS 22 760.25 786.45 772.20 1.39Crude fibre 22 152.00 172.62 163.44 1.34Crude fat 22 42.35 50.63 45.46 0.52Starch 22 190.65 249.79 219.59 3.49Sucrose 22 18.76 37.02 31.04 0.89Calcium 22 6.11 9.19 7.71 0.13Phosphorus 22 3.43 4.33 3.91 0.04Sodium 22 1.13 1.95 1.64 0.04Magnesium 22 2.14 2.75 2.41 0.03Potassium 22 15.33 18.44 16.74 0.17Sulphur 22 1.82 2.25 2.05 0.02Chloride 22 4.72 6.75 5.88 0.09Copper

mg/

kg D

M 22 20.48 30.89 26.14 0.55

Zinc 22 91.77 156.56 126.67 3.12Manganese 22 87.05 123.93 110.58 2.06Iron 22 294.80 403.26 355.99 6.72Selenium 22 0.55 0.88 0.70 0.02

Farm

8

Dry matter g/kg 18 384.37 455.96 425.43 5.43Crude ash

g/kg

DM

18 59.40 77.39 66.54 1.28Crude protein 18 164.83 215.36 180.37 2.97ELOS 18 747.41 790.46 765.91 2.78Crude fibre 18 139.60 169.19 156.89 1.79Crude fat 18 35.42 43.62 39.98 0.68Starch 18 233.01 282.87 256.73 3.37Sucrose 18 41.32 65.13 55.30 1.49Calcium 18 4.62 7.03 5.79 0.17Phosphorus 18 3.69 4.96 4.24 0.06Sodium 18 1.38 4.02 2.46 0.17Magnesium 18 2.11 2.73 2.30 0.04Potassium 18 11.55 15.12 13.17 0.26Sulphur 18 1.99 2.95 2.27 0.05Chloride 18 3.48 8.67 5.66 0.34Copper

mg/

kg D

M 18 12.98 36.35 23.26 1.81

Zinc 18 48.74 171.39 107.47 9.30Manganese 18 56.66 113.58 79.71 4.90Iron 18 317.19 921.56 417.01 31.57Selenium 18 0.33 1.32 0.78 0.07

89

Farm

9

Nutrients N Minimum Maximum Mean SEM Dry matter g/kg 20 307.22 375.96 356.26 4.54Crude ash

g/kg

DM

20 55.64 73.71 66.02 1.32Crude protein 20 138.39 181.30 161.28 2.44ELOS 20 722.05 774.15 749.16 3.19Crude fibre 20 151.85 192.40 164.69 2.44Crude fat 20 43.97 59.26 51.57 0.89Starch 20 217.25 284.21 252.81 3.70Sucrose 20 9.08 24.71 18.05 0.83Calcium 20 3.81 11.30 8.44 0.50Phosphorus 20 2.96 3.89 3.48 0.05Sodium 20 0.32 1.24 0.84 0.07Magnesium 20 1.58 2.31 2.00 0.05Potassium 20 9.66 11.40 10.32 0.10Sulphur 20 1.89 2.22 2.05 0.02Chloride 20 2.22 4.18 3.30 0.14Copper

mg/

kg D

M 20 9.08 29.96 20.43 1.58

Zinc 20 40.13 123.56 85.21 6.49Manganese 20 32.37 86.54 63.39 4.47Iron 20 429.62 657.99 517.34 14.81Selenium 20 0.05 0.73 0.43 0.05

Farm

10

Dry matter g/kg 20 390.95 475.12 428.85 4.71Crude ash

g/kg

DM

20 56.07 86.16 70.99 1.99Crude protein 20 155.83 197.23 174.23 1.99ELOS 20 742.12 791.87 772.42 2.53Crude fibre 20 156.77 189.82 168.27 1.87Crude fat 20 38.16 61.20 47.79 1.54Starch 20 200.04 247.60 234.08 2.36Sucrose 20 31.53 47.19 39.54 0.82Calcium 20 5.55 8.03 6.88 0.16Phosphorus 20 4.05 5.45 4.48 0.09Sodium 20 1.63 9.57 4.71 0.58Magnesium 20 2.03 2.46 2.22 0.03Potassium 20 11.34 13.56 12.41 0.15Sulphur 20 1.92 2.34 2.08 0.02Chloride 20 4.38 19.45 10.26 1.11Copper

mg/

kg D

M 20 13.76 28.57 21.82 0.97

Zinc 20 69.72 124.47 99.77 4.07Manganese 20 62.63 93.67 78.32 2.23Iron 20 377.82 520.61 459.29 9.07Selenium 20 0.29 0.85 0.58 0.03

90

Farm

11

Nutrients N Minimum Maximum Mean SEM

Dry matter g/kg 20 455.20 489.96 477.78 2.03

Crude ash

g/kg

DM

20 57.58 72.20 64.33 0.90

Crude protein 20 137.96 159.39 151.91 1.26

ELOS 20 711.37 769.80 746.57 3.39

Crude fibre 20 153.37 180.19 165.47 1.99

Crude fat 20 35.14 52.58 44.98 1.25

Starch 20 220.99 271.67 253.04 3.35

Sucrose 20 24.96 36.01 31.96 0.56

Calcium 20 5.65 9.23 7.18 0.20

Phosphorus 20 3.52 4.39 3.92 0.06

Sodium 20 0.86 3.35 1.70 0.18

Magnesium 20 2.03 2.65 2.34 0.04

Potassium 20 11.97 15.25 13.55 0.16

Sulphur 20 1.81 2.03 1.95 0.01

Chloride 20 2.30 4.30 3.18 0.10

Copper

mg/

kg D

M

20 9.94 39.05 22.39 1.50

Zinc 20 45.61 145.38 92.04 5.59

Manganese 20 31.80 82.40 54.12 2.80

Iron 20 191.87 290.12 238.66 6.44

Selenium 20 0.12 0.62 0.33 0.03

91

APPENDIX 3 Saxonian Dairy Herds Data – Trace Elements,

Health and Production Parameters

Farm

1

Parameters N Minimum Maximum Mean SEM Liver Trace Elements (Fresh matter basis)

Zinc (m/kg ) 10 6.97 41.70 23.37 3.36Copper (m/kg) 10 16.30 199.00 97.47 19.19Manganese (m/kg) 10 0.29 13.80 4.10 1.21Iron (m/kg) 10 30.30 278.00 101.53 24.72Selenium (μg/kg) 10 248.30 887.50 609.86 65.04

Plasma Trace Elements Zinc (mg/l) 8 1.32 2.01 1.63 0.07Copper (mg/l) 10 0.18 1.13 0.65 0.08Manganese (mg/l) 0 Iron (m/l) 8 0.93 2.53 1.86 0.19Selenium (μg/l) 10 89.24 108.60 95.80 1.94

Plasma Biochemistry ALAT (μkat/l) 10 0.58 0.92 0.74 0.04ASAT (μkat/l) 10 1.20 3.20 1.75 0.19Bilirubin (μmol/l) 10 2.20 4.90 4.09 0.25Bilirubin-direct 10 0.00 0.20 0.13 0.02Bilirubin indirect 10 2.20 4.80 3.98 0.24Ferritin (ng/ml) 10 7.10 76.50 36.13 6.49

Production Parameters December 2008 Milk yield (kg/day) 10 25.70 43.10 34.98 2.02Fat (%) 10 3.56 4.73 4.27 0.11Protein (%) 10 3.28 4.03 3.56 0.07Milk urea (mg/l) 10 270.00 430.00 331.00 17.41Somatic cell count (* 1000) 10 43.00 2162.00 318.70 205.94Lactose (%) 10 4.52 5.17 4.86 0.06Lactation number 10 1.00 6.00 3.10 0.53Days in milk 10 113.00 258.00 176.70 11.66Protein corrected milk (kg/day)

10 26.94 45.74 36.37 1.94

Production Parameters November 2008 Milk yield (kg/day) 10 27.60 43.60 35.08 1.45Fat (%) 10 3.32 4.39 3.89 0.12Protein (%) 10 2.65 3.86 3.39 0.10Milk urea (mg/l) 10 160.00 380.00 266.00 21.66Somatic cell count (* 1000) 10 26.00 3426.00 610.80 354.24Lactose (%) 10 4.43 5.17 4.83 0.06Lactation number 10 1.00 6.00 3.10 0.53Days in milk 10 78.00 223.00 141.70 11.66Protein corrected milk (kg/day)

10 26.60 39.85 34.57 1.33

92

Farm

2

Parameters N Minimum Maximum Mean SEM Liver Trace Elements (Fresh matter basis)

Zinc (m/kg ) 9 11.60 25.70 16.44 1.64Copper (m/kg) 9 34.40 265.00 137.40 24.09Manganese (m/kg) 10 2.81 8.28 5.48 0.54Iron (m/kg) 10 2.85 250.00 110.21 22.85Selenium (μg/kg) 9 255.70 793.80 547.74 58.79

Plasma Trace Elements Zinc (mg/l) 10 1.63 2.77 2.00 0.10Copper (mg/l) 10 0.53 4.14 1.77 0.38Manganese (mg/l) 1 0.02 0.02 0.02 .

Iron (m/l) 7 0.64 2.35 1.62 0.20Selenium (μg/l) 10 81.58 110.60 93.63 2.99

Plasma Biochemistry ALAT (μkat/l) 10 0.68 1.03 0.91 0.03ASAT (μkat/l) 10 1.36 2.16 1.79 0.08Bilirubin (μmol/l) 10 3.30 4.90 4.24 0.14Bilirubin-direct 10 0.00 0.20 0.10 0.02Bilirubin-indirect 10 3.30 4.80 4.14 0.14Ferritin (ng/ml) 10 13.80 64.40 36.65 6.26

Production Parameters December 2008 Milk yield (kg/day) 10 32.40 44.50 37.81 1.17Fat (%) 10 3.86 4.68 4.21 0.09Protein (%) 10 3.16 3.78 3.43 0.06Milk urea (mg/l) 10 200.00 310.00 243.00 11.93Somatic cell count (* 1000) 10 26.00 127.00 62.20 8.76Lactose (%) 10 4.45 4.85 4.69 0.05Lactation number 10 2.00 3.00 2.40 0.16Days in milk 10 121.00 195.00 167.80 7.82Protein corrected milk (kg/day)

10 34.10 47.74 38.86 1.30

Production Parameters November 2008 Milk yield (kg/day) 10 32.20 59.30 42.91 2.50Fat (%) 10 3.69 5.01 4.17 0.15Protein (%) 10 2.90 3.79 3.26 0.07Milk urea (mg/l) 10 200.00 330.00 277.00 13.34Somatic cell count (* 1000) 10 36.00 2943.00 340.60 289.19Lactose (%) 10 4.22 4.93 4.76 0.07Lactation number 10 2.00 3.00 2.40 0.16Days in milk 10 72.00 146.00 118.80 7.82Protein corrected milk (kg/day)

10 35.29 64.20 43.36 2.68

93

Farm

3

Parameters N Minimum Maximum Mean SEM Liver Trace Elements (Fresh matter basis)

Zinc (m/kg ) 10 11.60 23.20 20.94 1.08Copper (m/kg) 10 139.00 234.00 188.50 9.08Manganese (m/kg) 10 6.58 52.50 13.89 4.43Iron (m/kg) 9 4.34 56.10 30.97 5.37Selenium (μg/kg) 10 483.00 850.40 705.66 40.95

Plasma Trace Elements Zinc (mg/l) 9 0.69 2.43 1.71 0.21Copper (mg/l) 10 0.61 3.63 1.52 0.29Manganese (mg/l) 2 0.22 3.64 1.93 1.71Iron (m/l) 3 0.74 3.01 1.93 0.66Selenium (μg/l) 10 92.40 152.20 109.28 6.25

Plasma Biochemistry ALAT (μkat/l) 10 0.55 0.91 0.80 0.03ASAT (μkat/l) 10 1.00 3.03 2.00 0.19Bilirubin (μmol/l) 10 3.20 5.00 4.23 0.17Bilirubin-direct 10 0.00 0.30 0.11 0.03Bilirubin indirect 10 3.00 4.90 4.10 0.17Ferritin (ng/ml) 10 3.80 31.00 17.23 3.03

Production Parameters December 2008 Milk yield (kg/day) 10 29.20 44.00 36.23 1.61Fat (%) 10 3.55 5.89 4.28 0.22Protein (%) 10 3.28 3.96 3.55 0.06Milk urea (mg/l) 10 250.00 320.00 281.00 6.57Somatic cell count (* 1000) 10 24.00 227.00 80.80 21.17Lactose (%) 10 4.22 5.06 4.64 0.08Lactation number 10 1.00 6.00 2.90 0.46Days in milk 10 160.00 192.00 167.50 2.85Protein corrected milk (kg/day)

10 31.37 43.40 37.51 1.18

Production Parameters November 2008 Milk yield (kg/day) 10 27.00 45.00 38.08 1.66Fat (%) 10 3.65 5.23 4.30 0.17Protein (%) 10 3.21 3.95 3.49 0.07Milk urea (mg/l) 10 210.00 330.00 257.00 12.21Somatic cell count (* 1000) 10 19.00 349.00 84.00 30.68Lactose (%) 10 4.35 4.94 4.69 0.07Lactation number 10 1.00 6.00 2.90 0.46Days in milk 10 132.00 164.00 139.50 2.85Protein corrected milk (kg/day)

10 29.02 44.62 39.42 1.32

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Parameters N Minimum Maximum Mean SEM Liver Trace Elements (Fresh matter basis)

Zinc (m/kg ) 10 16.00 46.70 30.24 3.07Copper (m/kg) 10 87.10 279.00 150.97 16.98Manganese (m/kg) 10 4.45 28.30 16.54 2.60Iron (m/kg) 10 3.13 84.60 39.34 9.09Selenium (μg/kg) 10 461.50 1380.00 920.31 108.12

Plasma Trace Elements Zinc (mg/l) 8 0.65 2.01 1.29 0.17Copper (mg/l) 10 0.47 4.45 1.85 0.37Manganese (mg/l) 9 0.02 2.32 1.20 0.27Iron (m/l) 7 0.50 2.98 1.61 0.35Selenium (μg/l) 10 101.80 136.90 114.75 3.88

Plasma Biochemistry ALAT (μkat/l) 10 0.60 1.14 0.85 0.06ASAT (μkat/l) 10 0.89 2.74 1.59 0.19Bilirubin (μmol/l) 10 3.60 5.60 4.12 0.21Bilirubin-direct 10 0.00 0.50 0.16 0.05Bilirubin indirect 10 3.30 5.10 3.98 0.19Ferritin (ng/ml) 10 12.40 41.40 28.73 3.27

Production Parameters December 2008 Milk yield (kg/day) 10 25.50 47.40 33.43 2.28Fat (%) 10 3.33 5.10 3.79 0.17Protein (%) 10 3.30 3.99 3.63 0.07Milk urea (mg/l) 10 180.00 380.00 266.00 18.39Somatic cell count (* 1000) 10 9.00 234.00 59.20 20.78Lactose (%) 10 4.69 5.02 4.85 0.03Lactation number 10 1.00 2.00 1.40 0.16Days in milk 10 110.00 161.00 138.10 5.45Protein corrected milk (kg/day)

10 25.48 44.57 32.95 2.02

Production Parameters November 2008 Milk yield (kg/day) 10 26.10 44.70 33.75 2.09Fat (%) 10 2.64 4.55 3.63 0.18Protein (%) 10 2.97 3.72 3.41 0.07Milk urea (mg/l) 10 200.00 380.00 267.00 20.00Somatic cell count (* 1000) 10 9.00 919.00 120.20 89.01Lactose (%) 10 4.66 5.04 4.80 0.04Lactation number 10 1.00 2.00 1.40 0.16Days in milk 10 82.00 132.00 112.00 4.47Protein corrected milk (kg/day)

10 25.61 38.60 32.03 1.39

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Parameters N Minimum Maximum Mean SEM Liver Trace Elements (Fresh matter basis)

Zinc (m/kg ) 10 7.96 42.70 19.83 3.69Copper (m/kg) 9 1.39 211.00 134.28 20.87Manganese (m/kg) 10 6.52 23.50 13.09 1.83Iron (m/kg) 10 20.10 220.00 89.72 24.27Selenium (μg/kg) 10 202.70 857.50 520.04 63.06

Plasma Trace Elements Zinc (mg/l) 9 1.06 2.43 1.93 0.13Copper (mg/l) 10 0.61 3.35 1.61 0.26Manganese (mg/l) 4 0.05 0.43 0.19 0.09Iron (m/l) 7 0.72 2.22 1.51 0.19Selenium (μg/l) 10 59.40 98.17 82.45 4.14

Plasma Biochemistry ALAT (μkat/l) 10 0.62 1.04 0.88 0.04ASAT (μkat/l) 10 1.45 3.23 2.24 0.22Bilirubin (μmol/l) 10 2.20 5.80 4.26 0.33Bilirubin-direct 10 0.10 0.40 0.21 0.03Bilirubin indirect 10 2.00 5.60 4.03 0.32Ferritin (ng/ml) 10 14.20 161.40 43.00 13.79

Production Parameters December 2008 Milk yield (kg/day) 10 23.00 43.90 34.63 2.35Fat (%) 10 2.96 5.37 3.98 0.25Protein (%) 10 3.14 4.18 3.54 0.10Milk urea (mg/l) 10 210.00 290.00 253.00 10.33Somatic cell count (* 1000)

10 19.00 437.00 105.00 40.50

Lactose (%) 10 4.66 5.28 4.89 0.06Lactation number 10 1.00 3.00 1.90 0.31Days in milk 10 133.00 157.00 145.60 2.56Protein corrected milk (kg/day)

10 24.86 44.41 34.59 2.24

Production Parameters November 2008 Milk yield (kg/day) 10 23.90 46.30 35.10 2.13Fat (%) 10 2.71 4.79 3.82 0.23Protein (%) 10 3.02 3.99 3.36 0.10Milk urea (mg/l) 10 200.00 290.00 241.00 10.48Somatic cell count (* 1000)

10 16.00 641.00 146.70 62.84

Lactose (%) 10 4.49 5.13 4.85 0.06Lactation number 10 1.00 3.00 1.90 0.31Days in milk 10 98.00 122.00 110.60 2.56Protein corrected milk (kg/day)

10 26.17 41.75 33.92 1.69

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Parameters N Minimum Maximum Mean SEM Liver Trace Elements (Fresh matter basis)

Zinc (m/kg ) 9 9.28 23.00 16.63 1.80Copper (m/kg) 10 102.00 372.00 202.00 28.20Manganese (m/kg) 9 1.11 10.00 4.59 0.83Iron (m/kg) 10 10.60 255.00 81.54 28.93Selenium (μg/kg) 10 622.80 1566.00 970.57 113.82

Plasma Trace Elements Zinc (mg/l) 9 0.41 1.91 0.91 0.14Copper (mg/l) 9 0.89 7.68 2.39 0.68Manganese (mg/l) 6 0.02 4.20 1.30 0.66Iron (m/l) 6 0.24 1.88 1.22 0.24Selenium (μg/l) 9 66.95 118.70 93.98 5.45

Plasma Biochemistry ALAT (μkat/l) 10 0.69 0.89 0.78 0.02ASAT (μkat/l) 10 1.49 2.71 1.91 0.11Bilirubin (μmol/l) 10 1.60 5.70 3.77 0.37Bilirubin-direct 10 0.00 0.20 0.12 0.02Bilirubin indirect 10 0.00 5.40 3.48 0.47Ferritin (ng/ml) 10 8.40 45.70 23.45 4.43

Production Parameters December 2008 Milk yield (kg/day) 10 34.30 43.00 38.59 1.08Fat (%) 10 3.16 5.02 4.26 0.20Protein (%) 10 3.15 3.95 3.49 0.08Milk urea (mg/l) 10 250.00 300.00 277.00 5.78Somatic cell count (* 1000) 10 29.00 898.00 153.60 83.76Lactose (%) 10 4.67 4.93 4.82 0.03Lactation number 10 1.00 4.00 2.00 0.26Days in milk 10 91.00 197.00 133.80 11.55Protein corrected milk (kg/day)

10 32.83 46.04 39.89 1.11

Production Parameters November 2008 Milk yield (kg/day) 10 38.00 43.70 41.20 0.54Fat (%) 10 3.16 5.75 4.06 0.25Protein (%) 10 3.09 3.67 3.36 0.06Milk urea (mg/l) 10 200.00 310.00 246.00 9.09Somatic cell count (* 1000) 10 15.00 452.00 135.00 50.08Lactose (%) 10 4.74 5.03 4.89 0.03Lactation number 10 1.00 4.00 2.00 0.26Days in milk 10 56.00 162.00 98.80 11.55Protein corrected milk (kg/day)

10 34.78 52.87 41.46 1.59

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Parameters N Minimum Maximum Mean SEM Liver Trace Elements (Fresh matter basis)

Zinc (m/kg ) 10 2.40 9.72 6.11 0.76Copper (m/kg) 10 48.30 141.00 79.77 10.27Manganese (m/kg) 9 2.28 15.60 6.39 1.38Iron (m/kg) 10 52.70 187.00 129.98 14.71Selenium (μg/kg) 10 444.70 1049.00 720.80 58.44

Plasma Trace Elements Zinc (mg/l) 10 0.57 2.05 1.36 0.19Copper (mg/l) 10 0.34 8.09 2.41 0.81Manganese (mg/l) 5 0.08 1.04 0.34 0.18Iron (m/l) 7 0.09 6.07 2.61 0.74Selenium (μg/l) 10 94.03 137.80 113.20 4.85

Plasma Biochemistry ALAT (μkat/l) 10 0.84 1.18 0.93 0.03ASAT (μkat/l) 10 1.14 3.26 1.90 0.20Bilirubin (μmol/l) 10 2.70 6.70 4.36 0.34Bilirubin-direct 10 0.00 0.30 0.16 0.03Bilirubin indirect 10 2.60 6.40 4.21 0.31Ferritin (ng/ml) 10 12.10 88.40 38.02 8.44

Production Parameters December 2008 Milk yield (kg/day) 10 33.80 54.90 43.66 2.68Fat (%) 10 3.03 4.46 3.62 0.15Protein (%) 10 2.92 3.95 3.25 0.10Milk urea (mg/l) 10 200.00 320.00 263.00 12.12Somatic cell count (* 1000) 10 16.00 204.00 64.60 18.32Lactose (%) 10 4.40 5.08 4.83 0.06Lactation number 10 1.00 5.00 3.00 0.47Days in milk 10 102.00 173.00 134.80 7.87Protein corrected milk (kg/day)

10 31.01 50.26 41.07 2.09

Production Parameters November 2008 Milk yield (kg/day) 10 34.40 58.40 45.86 2.66Fat (%) 10 2.89 4.26 3.49 0.15Protein (%) 10 2.67 3.61 3.10 0.09Milk urea (mg/l) 10 180.00 290.00 245.00 10.25Somatic cell count (* 1000) 10 15.00 206.00 56.30 17.88Lactose (%) 10 4.34 5.09 4.83 0.06Lactation number 10 1.00 5.00 3.00 0.47Days in milk 10 76.00 157.00 117.20 8.60Protein corrected milk (kg/day)

10 31.66 50.71 42.12 2.29

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Parameters N Minimum Maximum Mean SEM Liver Trace Elements (Fresh matter basis)

Zinc (m/kg ) 10 6.19 31.30 15.30 2.54Copper (m/kg) 10 35.20 348.00 122.84 32.09Manganese (m/kg) 10 0.38 8.75 2.63 0.82Iron (m/kg) 10 12.30 273.00 125.24 24.03Selenium (μg/kg) 10 472.10 3322.00 1470.47 295.86

Plasma Trace Elements Zinc (mg/l) 10 0.39 1.54 0.87 0.10Copper (mg/l) 10 0.80 4.26 2.13 0.37Manganese (mg/l) 8 0.03 1.21 0.59 0.15Iron (m/l) 6 0.46 2.40 0.97 0.32Selenium (μg/l) 10 99.67 167.60 118.76 6.22

Plasma Biochemistry ALAT (μkat/l) 10 0.62 1.01 0.82 0.04ASAT (μkat/l) 10 1.17 3.08 2.21 0.19Bilirubin (μmol/l) 10 1.40 6.00 4.34 0.41Bilirubin-direct 10 0.00 0.40 0.19 0.04Bilirubin indirect 10 1.30 5.80 4.11 0.40Ferritin (ng/ml) 10 16.20 38.50 26.52 2.62

Production Parameters December 2008 Milk yield (kg/day) 10 20.20 48.90 35.66 2.99Fat (%) 10 2.63 4.76 3.53 0.22Protein (%) 10 3.01 3.90 3.48 0.08Milk urea (mg/l) 10 190.00 340.00 276.00 16.75Somatic cell count (* 1000)

10 29.00 277.00 118.10 27.51

Lactose (%) 10 4.29 4.94 4.71 0.07Lactation number 10 1.00 4.00 1.90 0.38Days in milk 10 120.00 205.00 144.50 7.20Protein corrected milk (kg/day)

10 19.15 45.28 33.55 2.48

Production Parameters November 2008 Milk yield (kg/day) 10 20.60 49.70 37.38 2.80Fat (%) 10 2.67 4.92 3.57 0.21Protein (%) 10 3.15 3.76 3.45 0.07Milk urea (mg/l) 10 180.00 310.00 238.00 14.44Somatic cell count (* 1000)

10 16.00 3010.00 355.90 295.38

Lactose (%) 10 4.35 5.08 4.84 0.07Lactation number 10 1.00 4.00 1.90 0.38Days in milk 10 85.00 170.00 109.50 7.20Protein corrected milk (kg/day)

10 20.93 49.19 35.44 2.64

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Parameters N Minimum Maximum Mean SEM Liver Trace Elements (Fresh matter basis)

Zinc (m/kg ) 9 13.00 32.00 20.57 2.16Copper (m/kg) 9 69.70 152.00 121.19 7.96Manganese (m/kg) 9 0.52 11.70 3.18 1.18Iron (m/kg) 9 21.50 150.89 76.10 15.49Selenium (μg/kg) 9 424.90 828.40 554.01 42.13

Plasma Trace Elements Zinc (mg/l) 9 0.60 2.01 1.47 0.17Copper (mg/l) 9 0.24 5.21 1.63 0.57Manganese (mg/l) 6 0.02 0.14 0.07 0.02Iron (m/l) 7 0.99 4.67 1.94 0.46Selenium (μg/l) 9 54.73 123.70 82.12 7.45

Plasma Biochemistry ALAT (μkat/l) 10 0.68 1.15 0.89 0.04ASAT (μkat/l) 10 1.15 2.37 1.82 0.14Bilirubin (μmol/l) 10 1.70 5.10 3.95 0.38Bilirubin-direct 10 0.00 0.20 0.08 0.02Bilirubin indirect 10 1.70 5.10 4.09 0.30Ferritin (ng/ml) 10 10.50 69.20 31.19 6.10

Production Parameters December 2008 Milk yield (kg/day) 10 22.90 39.90 33.31 1.46Fat (%) 10 2.77 4.80 3.86 0.19Protein (%) 10 3.07 3.69 3.42 0.07Milk urea (mg/l) 10 140.00 230.00 191.00 9.36Somatic cell count (* 1000)

10 21.00 235.00 84.60 24.54

Lactose (%) 10 4.52 4.96 4.78 0.05Lactation number 10 1.00 5.00 2.50 0.43Days in milk 10 133.00 321.00 187.10 17.24Protein corrected milk (kg/day)

10 22.45 38.35 32.76 1.47

Production Parameters November 2008 Milk yield (kg/day) 10 24.90 42.10 32.51 1.49Fat (%) 10 2.93 4.73 3.97 0.18Protein (%) 10 3.22 3.70 3.44 0.05Milk urea (mg/l) 10 130.00 220.00 183.00 10.65Somatic cell count (* 1000)

10 21.00 221.00 100.90 21.67

Lactose (%) 10 4.59 4.98 4.84 0.04Lactation number 10 1.00 5.00 2.50 0.43Days in milk 10 109.00 297.00 163.10 17.24Protein corrected milk (kg/day)

10 24.91 39.09 32.38 1.24

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Parameters N Minimum Maximum Mean SEM Liver Trace Elements (Fresh matter basis)

Zinc (m/kg ) 9 14.10 25.70 19.62 1.34Copper (m/kg) 9 66.60 227.00 130.36 18.68Manganese (m/kg) 10 2.55 12.50 4.41 0.93Iron (m/kg) 10 29.20 156.00 87.18 13.64Selenium (μg/kg) 9 576.90 1508.00 904.90 111.30

Plasma Trace Elements Zinc (mg/l) 5 1.17 2.54 1.79 0.23Copper (mg/l) 10 1.03 5.27 2.09 0.44Manganese (mg/l) 9 0.05 2.30 1.24 0.28Iron (m/l) 4 0.99 4.67 1.94 0.46Selenium (μg/l) 10 94.96 138.20 113.40 4.40

Plasma Biochemistry ALAT (μkat/l) 10 0.63 1.10 0.89 0.04ASAT (μkat/l) 10 1.12 2.01 1.51 0.07Bilirubin (μmol/l) 10 3.20 4.70 4.10 0.16Bilirubin-direct 10 0.00 0.20 0.11 0.02Bilirubin indirect 10 3.10 4.70 4.02 0.15Ferritin (ng/ml) 10 15.20 66.70 31.53 5.03

Production Parameters December 2008 Milk yield (kg/day) 10 22.00 41.90 33.63 1.94Fat (%) 10 3.24 5.05 3.98 0.17Protein (%) 10 3.09 3.53 3.30 0.05Milk urea (mg/l) 10 180.00 270.00 236.00 7.77Somatic cell count (* 1000) 10 4.92 2417.00 388.69 236.80Lactose (%) 10 4.36 4.96 4.70 0.05Lactation number 10 1.00 6.00 2.90 0.62Days in milk 0 Protein corrected milk (kg/day)

10 24.65 42.70 33.23 1.83

Production Parameters November 2008 Milk yield (kg/day) 10 26.10 46.30 36.12 2.19Fat (%) 10 2.83 3.91 3.49 0.12Protein (%) 10 3.17 3.82 3.47 0.06Milk urea (mg/l) 10 190.00 300.00 258.00 11.23Somatic cell count (* 1000) 10 36.00 1689.00 399.10 195.76Lactose (%) 10 4.44 5.06 4.78 0.06Lactation number 10 1.00 6.00 2.90 0.62Days in milk 0 Protein corrected milk (kg/day)

10 25.41 43.93 34.02 1.91

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Parameters N Minimum Maximum Mean SEM Liver Trace Elements (Fresh matter basis)

Zinc (m/kg ) 10 1.49 21.40 11.83 1.95Copper (m/kg) 9 21.90 205.00 111.87 21.89Manganese (m/kg) 9 1.46 9.19 4.09 0.75Iron (m/kg) 10 40.89 204.00 101.92 16.78Selenium (μg/kg) 10 122.60 1127.00 595.72 98.66

Plasma Trace Elements Zinc (mg/l) 7 1.53 2.33 1.91 0.11Copper (mg/l) 8 0.26 6.37 1.64 0.69Manganese (mg/l) 4 0.06 1.39 0.56 0.30Iron (m/l) 4 2.25 3.34 2.74 0.23Selenium (μg/l) 8 85.79 104.00 96.57 2.11

Plasma Biochemistry ALAT (μkat/l) 10 0.67 0.96 0.80 0.03ASAT (μkat/l) 10 1.34 2.54 2.08 0.13Bilirubin (μmol/l) 10 1.50 8.10 4.25 0.53Bilirubin-direct 10 0.00 0.20 0.08 0.02Bilirubin indirect 10 0.00 7.80 4.06 0.61Ferritin (ng/ml) 10 11.70 48.40 28.27 4.70

Production Parameters December 2008 Milk yield (kg/day) 10 25.80 42.40 35.28 2.01Fat (%) 10 2.80 5.19 3.66 0.25Protein (%) 10 2.81 4.17 3.34 0.12Milk urea (mg/l) 10 160.00 270.00 218.00 10.52Somatic cell count (* 1000) 10 22.00 1980.00 313.70 193.12Lactose (%) 10 4.39 5.03 4.78 0.06Lactation number 10 1.00 4.00 2.20 0.33Days in milk 10 112.00 177.00 144.20 7.26Protein corrected milk (kg/day)

10 24.14 41.56 33.48 1.70

Production Parameters November 2008 Milk yield (kg/day) 10 32.00 48.20 41.03 1.61Fat (%) 10 2.00 4.99 3.02 0.30Protein (%) 10 3.04 4.14 3.40 0.11Milk urea (mg/l) 10 150.00 270.00 223.00 11.65Somatic cell count (* 1000) 10 13.00 1141.00 171.40 110.26Lactose (%) 10 4.48 5.04 4.81 0.06Lactation number 10 1.00 4.00 2.20 0.33Days in milk 10 84.00 149.00 112.70 7.67Protein corrected milk (kg/day)

10 31.46 41.40 36.03 1.17